Plant amino acyl-tRNA synthetase

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding an aminoacyl-tRNA synthetase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the aminoacyl-tRNA synthetase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the aminoacyl-tRNA synthetase in a transformed host cell.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/092,866, filed Jul. 15, 1998.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingaminoacyl-tRNA synthetase in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] All tRNAs have two functions: to chemically link to a specificamino acid and to recognize a codon in mRNA so that the linked aminoacid can be added to a growing peptide chain during protein synthesis.In general there is at least one aminoacyl-tRNA synthetase for each ofthe twenty amino acids. A specific aminoacyl-tRNA synthetase links anamino acid to the 2′ or 3′ hydroxyl of the adenosine residue at the3′-terminus of a tRNA molecule. Once its correct amino acid is attached,a tRNA then recognizes a codon in mRNA, thus deliverng its amino acid tothe growing polypeptide chain. These enzymatic functions are critical togene expression (Neidhart et al. (1975) Annu. Rev. Microbiol.29:215-250). Mutations in tRNA synthetases often result in alterationsin protein synthesis and in some cases cell death.

[0004] Plants like other cellular organisms have aminoacyl-tRNAsynthetases. However a complete description of the plant ‘complement’ ofaminoacyl-tRNA synthetases has not been published. It is anticipatedthat plants will likely have at least forty aminoacyl-tRNA synthetases.Plants have three sites of protein synthesis: the cytoplasm, themitochondria and the chloroplast. Accordingly, there could be as many assixty aminoacyl-tRNA synthetases. Based on knowledge of other eukaryotesthe cytoplasmic and mitochondrial aminoacyl-tRNA synthetases areexpected to be encoded by the same gene. This gene should be nuclearlyencoded and produce two alternate products, one with a mitochondrialspecific transit peptide, and the other without this targeting signal.The chloroplast is the other site of protein synthesis in plants. Basedon a few examples of known plant chloroplast specific aminoacyl-tRNAsynthetase genes it appears that these genes are also nuclear-encoded.Chloroplast aminoacyl-tRNA synthetases are directed to the chloroplastby a transit peptide.

[0005] Because of the central role aminoacyl-tRNA synthetases play inprotein synthesis any agent that inhibits or disrupts aminoacyl-tRNAsynthetase activity is likely to be toxic. Indeed a number ofaminoacyl-tRNA synthetase inhibitors (antibiotics and herbicides) areknown (Zon et al. (1988) Phytochemistry 27(3):711-714 and Heacock et al.(1996) Bioorganic Chemistry 24(3):273-289). Thus it may be possible todevelop new herbicides that target aminoacyl-tRNA synthetases andengineer aminoacyl-tRNA synthetases that are resistant to suchherbicides. Accordingly, the availability of nucleic acid sequencesencoding all or a portion of these enzymes would facilitate studies tobetter understand protein synthesis in plants, provide genetic tools forthe manipulation of gene expression, and provide a possible target forherbicides.

SUMMARY OF THE INVENTION

[0006] The instant invention relates to isolated nucleic acid fragmentsencoding aminoacyl-tRNA synthetase. Specifically, this inventionconcerns an isolated nucleic acid fragment encoding an aspartyl-tRNAsynthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase ortyrosyl-tRNA synthetase and an isolated nucleic acid fragment that issubstantially similar to an isolated nucleic acid fragment encoding anaspartyl-tRNA synthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNAsynthetase or tyrosyl-tRNA synthetase. In addition, this inventionrelates to a nucleic acid fragment that is complementary to the nucleicacid fragment encoding aspartyl-tRNA synthetase, cysteinyl-tRNAsynthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase.

[0007] An additional embodiment of the instant invention pertains to apolypeptide encoding all or a substantial portion of an aminoacyl-tRNAsynthetaseselected from the group consisting of aspartyl-tRNAsynthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase andtyrosyl-tRNA synthetase.

[0008] In another embodiment, the instant invention relates to achimeric gene encoding an aspartyl-tRNA synthetase, cysteinyl-tRNAsynthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase, orto a chimeric gene that comprises a nucleic acid fragment that iscomplementary to a nucleic acid fragment encoding an aspartyl-tRNAsynthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase ortyrosyl-tRNA synthetase, operably linked to suitable regulatorysequences, wherein expression of the chimeric gene results in productionof levels of the encoded protein in a transformed host cell that isaltered (i.e., increased or decreased) from the level produced in anuntransformed host cell.

[0009] In a further embodiment, the instant invention concerns atransformed host cell comprising in its genome a chimeric gene encodingan aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase, operably linkedto suitable regulatory sequences. Expression of the chimeric generesults in production of altered levels of the encoded protein in thetransformed host cell. The transformed host cell can be of eukaryotic orprokaryotic origin, and include cells derived from higher plants andmicroorganisms. The invention also includes transformed plants thatarise from transformed host cells of higher plants, and seeds derivedfrom such transformed plants.

[0010] An additional embodiment of the instant invention concerns amethod of altering the level of expression of an aspartyl-tRNAsynthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase ortyrosyl-tRNA synthetase in a transformed host cell comprising: a)transforming a host cell with a chimeric gene comprising a nucleic acidfragment encoding an aspartyl-tRNA synthetase, cysteinyl-tRNAsynthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase; andb) growing the transformed host cell under conditions that are suitablefor expression of the chimeric gene wherein expression of the chimericgene results in production of altered levels of aspartyl-tRNAsynthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase ortyrosyl-tRNA synthetase in the transformed host cell.

[0011] An addition embodiment of the instant invention concerns a methodfor obtaining a nucleic acid fragment encoding all or a substantialportion of an amino acid sequence encoding an aspartyl-tRNA synthetase,cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNAsynthetase.

[0012] A further embodiment of the instant invention is a method forevaluating at least one compound for its ability to inhibit the activityof an aspartyl-tRNA synthetase, cysteinyl-tRNA synthetase,tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase, the methodcomprising the steps of: (a) transforming a host cell with a chimericgene comprising a nucleic acid fragment encoding an aspartyl-tRNAsynthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase ortyrosyl-tRNA synthetase, operably linked to suitable regulatorysequences; (b) growing the transformed host cell under conditions thatare suitable for expression of the chimeric gene wherein expression ofthe chimeric gene results in production of aspartyl-tRNA synthetase,cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNAsynthetase in the transformed host cell; (c) optionally purifying theaspartyl-tRNA synthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNAsynthetase or tyrosyl-tRNA synthetase expressed by the transformed hostcell; (d) treating the aspartyl-tRNA synthetase, cysteinyl-tRNAsynthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNA synthetase witha compound to be tested; and (e) comparing the activity of theaspartyl-tRNA synthetase, cysteinyl-tRNA synthetase, tryptophanyl-tRNAsynthetase or tyrosyl-tRNA synthetase that has been treated with a testcompound to the activity of an untreated aspartyl-tRNA synthetase,cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNAsynthetase, thereby selecting compounds with potential for inhibitoryactivity.

BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS

[0013] The invention can be more fully understood from the followingdetailed description and the accompanying Sequence Listing which form apart of this application.

[0014] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Aninoacyl-tRNA Synthetase SEQID NO: (Amino Protein Clone Designation (Nucleotide) Acid) Aspartyl-tRNASynthetase p0094.cssth73r 1 2 Aspartyl-tRNA Synthetase rl0n.pk0015.g11 34 Aspartyl-tRNA Synthetase sfl1.pk0046.e8 5 6 Aspartyl-tRNA Synthetasewle1n.pk0021.e6 7 8 Cysteinyl-tRNA Synthetase p0119.cmtmt52r 9 10Cysteinyl-tRNA Synthetase rsl1n.pk016.p18 11 12 Cysteinyl-tRNASynthetase sfl1.pk0013.f9 13 14 Tryptophanyl-tRNA p0118.chsbl87r 15 16Synthetase Tryptophanyl-tRNA sdp4c.pk033.n11 17 18 SynthetaseTryptophanyl-tRNA wlm4.pk0013.c12 19 20 Synthetase Tyrosyl-tRNASynthetase cs1.pk0035.d2 21 22

[0015] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0016] In the context of this disclosure, a number of terms shall beutilized. As used herein, a “nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. A nucleic acidfragment in the form of a polymer of DNA may be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA.

[0017] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof.

[0018] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by nucleic acid fragments that do not share 100%sequence identity with the gene to be suppressed. Moreover, alterationsin a nucleic acid fragment which result in the production of achemically equivalent amino acid at a given site, but do not effect thefunctional properties of the encoded polypeptide, are well known in theart. Thus, a codon for the amino acid alanine, a hydrophobic amino acid,may be substituted by a codon encoding another less hydrophobic residue,such as glycine, or a more hydrophobic residue, such as valine, leucine,or isoleucine. Similarly, changes which result in substitution of onenegatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts.

[0019] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6× SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2× SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2× SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2× SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1× SSC, 0.1% SDS at 65°C.

[0020] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Preferred are those nucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 80% identical to theamino acid sequences reported herein. More preferred nucleic acidfragments encode amino acid sequences that are 90% identical to theamino acid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% identical to theamino acid sequences reported herein. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0021] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0022] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0023] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0024] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0025] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0026] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic nucleotide segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. Promoters which cause a nucleic acid fragmentto be expressed in most cell types at most times are commonly referredto as “constitutive promoters”. New promoters of various types useful inplant cells are constantly being discovered; numerous examples may befound in the compilation by Okamuro and Goldberg (1989) Biochemistry ofPlants 15:1-82. It is further recognized that since in most cases theexact boundaries of regulatory sequences have not been completelydefined, nucleic acid fragments of different lengths may have identicalpromoter activity.

[0027] The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) MolecularBiotechnology 3:225).

[0028] The “3′ non-coding sequences” refer to nucleotide sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. The use of different 3′ non-coding sequences is exemplifiedby Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0029] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0030] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single nucleic acid fragment so thatthe function of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0031] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0032] “Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

[0033] “Mature” protein refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

[0034] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

[0035] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms. Examples ofmethods of plant transformation include Agrobacterium-mediatedtransformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

[0036] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0037] Nucleic acid fragments encoding at least a portion of severalaninoacyl-tRNA synthetases have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

[0038] For example, genes encoding other aspartyl-tRNA synthetase,cysteinyl-tRNA synthetase, tryptophanyl-tRNA synthetase or tyrosyl-tRNAsynthetase enzymes, either as cDNAs or genomic DNAs, could be isolateddirectly by using all or a portion of the instant nucleic acid fragmentsas DNA hybridization probes to screen libraries from any desired plantemploying methodology well known to those skilled in the art. Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis).Moreover, the entire sequences can be used directly to synthesize DNAprobes by methods known to the skilled artisan such as random primer DNAlabeling, nick translation, or end-labeling techniques, or RNA probesusing available in vitro transcription systems. In addition, specificprimers can be designed and used to amplify a part or all of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length cDNA or genomic fragmentsunder conditions of appropriate stringency.

[0039] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998) to generate cDNAs by using PCR to amplify copies of theregion between a single point in the transcript and the 3′ or 5′ end.Primers oriented in the 3′ and 5′ directions can be designed from theinstant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673; Loh et al. (1989)Science 243:217). Products generated by the 3′ and 5′ RACE procedurescan be combined to generate full-length cDNAs (Frohman and Martin (1989)Techniques 1:165).

[0040] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner (1984) Adv. Immunol. 36:1; Maniatis).

[0041] The nucleic acid fragments of the instant invention may be usedto create transgenic plants in which the disclosed polypeptides arepresent at higher or lower levels than normal or in cell types ordevelopmental stages in which they are not normally found. This wouldhave the effect of altering the level of aminoacyl-tRNA synthetaseactivity in those cells.

[0042] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

[0043] Plasmid vectors comprising the instant chimeric gene can thenconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

[0044] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

[0045] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

[0046] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0047] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppresiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different chimeric genes utilizing differentregulatory elements known to the skilled artisan. Once transgenic plantsare obtained by one of the methods described above, it will be necessaryto screen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds, and is notan inherent part of the invention. For example, one can screen bylooking for changes in gene expression by using antibodies specific forthe protein encoded by the gene being suppressed, or one could establishassays that specifically measure enzyme activity. A preferred methodwill be one which allows large numbers of samples to be processedrapidly, since it will be expected that a large number of transformantswill be negative for the desired phenotype.

[0048] The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded aninoacyl-tRNA synthetase. An example of a vector for highlevel expression of the instant polypeptides in a bacterial host isprovided (Example 9).

[0049] Additionally, the instant polypeptides can be used as a targetsto facilitate design and/or identification of inhibitors of thoseenzymes that may be useful as herbicides. This is desirable because thepolypeptides described herein catalyze various steps in proteinsynthesis. Accordingly, inhibition of the activity of one or more of theenzymes described herein could lead to inhibition plant growth. Thus,the instant polypeptides could be appropriate for new herbicidediscovery and design.

[0050] All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J Hum. Genet.32:314-331).

[0051] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bematzky and Tanksley (1986) Plant Mol.Biol. Reporter 4(1):37-41. Numerous publications describe geneticmapping of specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0052] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0053] In another embodiment, nucleic acid probes derived from theinstant nucleic acid sequences may be used in direct fluorescence insitu hybridization (FISH) mapping (Trask (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan et al. (1995) GenomeResearch 5:13-20), improvements in sensitivity may allow performance ofFISH mapping using shorter probes.

[0054] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997)Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) NucleicAcid Res. 1 7:6795-6807). For these methods, the sequence of a nucleicacid fragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0055] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al. (1995)Proc.Natl. Acad. Sci USA 92:8149; Bensen et al. (1995) Plant Cell 7:75). Thelatter approach may be accomplished in two ways. First, short segmentsof the instant nucleic acid fragments may be used in polymerase chainreaction protocols in conjunction with a mutation tag sequence primer onDNAs prepared from a population of plants in which Mutator transposonsor some other mutation-causing DNA element has been introduced (seeBensen, supra). The amplification of a specific DNA fragment with theseprimers indicates the insertion of the mutation tag element in or nearthe plant gene encoding the instant polypeptides. Alternatively, theinstant nucleic acid fragment may be used as a hybridization probeagainst PCR amplification products generated from the mutationpopulation using the mutation tag sequence primer in conjunction with anarbitrary genomic site primer, such as that for a restriction enzymesite-anchored synthetic adaptor. With either method, a plant containinga mutation in the endogenous gene encoding the instant polypeptides canbe identified and obtained. This mutant plant can then be used todetermine or confirm the natural function of the instant polypeptidesdisclosed herein.

EXAMPLES

[0056] The present invention is further defined in the followingExamples, in which all parts and percentages are by weight and degreesare Celsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

[0057] cDNA libraries representing mRNAs from various corn, rice,soybean and wheat tissues were prepared. The characteristics of thelibraries are described below. TABLE 2 cDNA Libraries from Corn, Rice,Soybean and Wheat Library Tissue Clone cs1 Corn leaf sheath from 5 weekold plant cs1.pk0035.d2 p0094 Corn ear leaf sheath, 2-3 p0094.cssth73rweeks after pollen shed* p0118 Corn pooled stem tissue fromp0118.chsbl87r the 4-5 internodes subtending the tassel, V8-V12 stages*p0119 Corn ear shoot/w husk: V-12 stage* p0119.cmtmt52r rl0n Rice 15 dayold leaf* rl0n.pk0015.g11 rsl1n Rice 15 day old seedling*rsl1n.pk016.p18 sdp4c Soybean developing embryo (9-11 mm)sdp4c.pk033.n11 sfl1 Soybean immature flower sfl1.pk0013.f9sfl1.pk0046.e8 wle1n Wheat leaf from 7 day wle1n.pk0021.e6 old etiolatedseedling* wlm4 Wheat seedlings 4 hours after wlm4.pk0013.c12 treatmentwith a fungicide**

[0058] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP* XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP* XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651). The resulting ESTs are analyzed using a Perkin Elmer Model377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

[0059] cDNA clones encoding aninoacyl-tRNA synthetases were identifiedby conducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/)searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) NatureGenetics 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Aspartyl-tRNASynthetase

[0060] The BLASTX search using the EST sequences from clones listed inTable 3 revealed similarity of the polypeptides encoded by the cDNAs toaspartyl-tRNA synthetase from Drosophila melanogaster (NCBI IdentifierNo. gi 4512034), Rattus norvegicus (NCBI Identifier No. gi 135099) andHomo sapiens (NCBI Identifier no. gi 4557513). Shown in Table 3 are theBLAST results for individual ESTs (“EST”), the sequences of the entirecDNA inserts comprising the indicated cDNA clones (“FIS”), or contigsassembled from two or more ESTs (“Contig”): TABLE 3 BLAST Results forSequences Encoding Polypeptides Homologous to Drosophila melanogaster,Rattus norvegicus and Homo sapiens Aspartyl-tRNA Synthetase Clone StatusBLAST pLog Score p0094.cssth73r FIS 134.00 (gi 4512034) rl0n.pk0015.g11FIS  51.15 (gi 135099) sfl1.pk0046.e8 FIS 102.00 (gi 4557513)wle1n.pk0021.e6 FIS  21.40 (gi 4557513)

[0061] The data in Table 4 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6and 8 and the Drosophila melanogaster, Rattus norvegicus and Homosapiens aspartyl-tRNA synthetase sequences (SEQ ID NOs: 23, 24 and 25respectively). TABLE 4 Percent Identity of Amino Acid Sequences DeducedFrom the Nucleotide Sequences of cDNA Clones Encoding PolypeptidesHomologous to Drosophila melanogaster, Rattus norvegicus and Homosapiens Aspartyl-tRNA Synthetase SEQ ID NO. Percent Identity to 2 51%(gi 4512034) 4 65% (gi 135099) 6 51% (gi 4557513) 8 52% (gi 4557513)

[0062] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151 -153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of an aspartyl-tRNAsynthetase. These sequences represent the first corn, rice, soybean andwheat sequences encoding aspartyl-tRNA synthetase.

Example 4 Characterization of cDNA Clones Encoding Cysteinyl-tRNASynthetase

[0063] The BLASTX search using the EST sequences from clones listed inTable 5 revealed similarity of the polypeptides encoded by the cDNAs tocysteinyl-tRNA synthetase from Haemophilus influenzae (NCBI IdentifierNo. gi 1174501) and Escherichia coli (NCBI Identifier No. gi 41203).Shown in Table 5 are the BLAST results for individual ESTs (“EST”), thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“FIS”), or contigs assembled from two or more ESTs (“Contig”):TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous toHaemophilus influenzae and Escherichia coli Cysteinyl-tRNA SynthetaseClone Status BLAST pLog Score p0119.cmtmt52r FIS 104.00 (gi 1174501)rsl1n.pk016.p18 FIS 108.00 (gi 41203) sfl1.pk0013.f9 FIS 117.00 (gi1174501)

[0064] The data in Table 6 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs: 10, 12 and14 and the Haemophilus influenzae and Escherichia coli sequences (SEQ IDNOs: 26 and 27 respectively). TABLE 6 Percent Identity of Amino AcidSequences Deduced From the Nucleotide Sequences of cDNA Clones EncodingPolypeptides Homologous to Haemophilus influenzae and Escherichia coliCysteinyl-tRNA Synthetase SEQ ID NO. Percent Identity to 10 43% (gi1174501) 12 44% (gi 41203) 14 44% (gi 1174501)

[0065] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of a cysteinyl-tRNAsynthetase. These sequences represent the first corn, rice and soybeansequences encoding cysteinyl-tRNA synthetase.

Example 5 Characterization of cDNA Clones Encoding Tryptophanyl-tRNASynthetase

[0066] The BLASTX search using the EST sequences from clones listed inTable 7 revealed similarity of the polypeptides encoded by the cDNAs totryptophanyl-tRNA synthetase from Synechocystis sp. (NCBI Identifier No.gi 2501072). Shown in Table 7 are the BLAST results for individual ESTs(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), or contigs assembled from two or moreESTs (“Contig”): TABLE 7 BLAST Results for Sequences EncodingPolypeptides Homologous to Synechocystis sp. Tryptophanyl-tRNASynthetase BLAST pLog Score Clone Status to (gi 2501072) p0118.chsbl87rEST 104.00 sdp4c.pk033.n11 FIS 103.00 wlm4.pk0013.c12 FIS  43.22

[0067] The data in Table 8 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs: 16, 18 and24 and the Synechocystis sp. sequence (SEQ ID NO: 28). TABLE 8 PercentIdentity of Amino Acid Sequences Deduced From the Nucleotide Sequencesof cDNA Clones Encoding Polypeptides Homologous to Synechocystis sp.Tryptophanyl-tRNA Synthetase Percent Identity to SEQ ID NO. (gi 2501072)16 49% 18 50% 20 51%

[0068] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of a tryptophanyl-tRNAsynthetase. These sequences represent the first corn, soybean and wheatsequences encoding tryptophanyl-tRNA synthetase.

Example 6 Characterization of cDNA Clones Encoding Tyrosyl-tRNASynthetase

[0069] The BLASTX search using the EST sequence from the clone listed inTable 9 revealed similarity of the polypeptide encoded by the cDNA totyrosyl-tRNA synthetase from Bacillus caldotenax (NCBI Identifier No. gi135196). Shown in Table 9 are the BLAST results for the sequence of theentire cDNA insert comprising the indicated cDNA clone (“FIS”): TABLE 9BLAST Results for Sequence Encoding Polypeptide Homologous to Bacilluscaldotenax Tyrosyl-tRNA Synthetase BLAST pLog Score to Clone Status (gi135196) cs1.pk0035.d2 FIS 62.52

[0070] The data in Table 10 represents a calculation of the percentidentity of the amino acid sequence set forth in SEQ ID NO:22 theBacillus caldotenax sequence (SEQ ID NO: 29). TABLE 10 Percent Identityof Amino Acid Sequence Deduced From the Nucleotide Sequence of cDNAClone Encoding Polypeptide Homologous to Bacillus caldotenaxTyrosyl-tRNA Synthetase Percent Identity to SEQ ID NO. (gi 135196) 2252%

[0071] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of a tyrosyl-tRNAsynthetase. This sequence represent the first corn sequence encodingtyrosyl-tRNA synthetase.

Example 7 Expression of Chimeric Genes in Monocot Cells

[0072] A chimeric gene comprising a cDNA encoding the instantpolypeptides in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and SmaI and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a chimeric gene encoding,in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNAfragment encoding the instant polypeptides, and the 10 kD zein 3′region.

[0073] The chimeric gene described above can then be introduced intocorn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0074] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35 S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0075] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0076] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0077] Seven days after bombardment the tissue can be transferred to N6medium that contains gluphosinate (2 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining gluphosinate. After 6 weeks, areas of about 1 cm in diameterof actively growing callus can be identified on some of the platescontaining the glufosinate-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0078] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 8 Expression of Chimeric Genes in Dicot Cells

[0079] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′ ) from thetranslation initiation codon and about 1650 nucleotides downstream (3′ )from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

[0080] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0081] Soybean embroys may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0082] Soybean embryogenic suspension cultures can maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0083] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0084] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0085] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0086] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0087] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 9 Expression of Chimeric Genes in Microbial Cells

[0088] The cDNAs' encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT 430.

[0089] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC).Buffer and agarose contain 10 μg/ml ethidium bromide for visualizationof the DNA fragment. The fragment can then be purified from the agarosegel by digestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0090] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J.Mol. Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25°. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

Example 10 Evaluating Compounds for Their Ability to Inhibit theActivity of Aminoacyl-tRNA Synthetase

[0091] The polypeptides described herein may be produced using anynumber of methods known to those skilled in the art. Such methodsinclude, but are not limited to, expression in bacteria as described inExample 9, or expression in eukaryotic cell culture, in planta, andusing viral expression systems in suitably infected organisms or celllines. The instant polypeptides may be expressed either as mature formsof the proteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆”). The fusion proteins may beengineered with a protease recognition site at the fusion point so thatfusion partners can be separated by protease digestion to yield intactmature enzyme. Examples of such proteases include thrombin, enterokinaseand factor Xa. However, any protease can be used which specificallycleaves the peptide connecting the fusion protein and the enzyme.

[0092] Purification of the instant polypeptides, if desired, may utilizeany number of separation technologies familiar to those skilled in theart of protein purification. Examples of such methods include, but arenot limited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose-4B. In an alternate embodiment,a thioredoxin fusion protein may be eluted using dithiothreitol;however, elution may be accomplished using other reagents which interactto displace the thioredoxin from the resin. These reagents includeβ-mercaptoethanol or other reduced thiol. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

[0093] Crude, partially purified or purified enzyme, either alone or asa fusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions which permit optimal enzymaticactivity. For example, assays for aminoacyl-tRNA synthetases arepresented by Zon et al. (1988) Phytochemistry 27(3):711-714 and Heacocket al. (1996) Bioorganic Chemistry 24(3):273-289.

1 29 1 1948 DNA Zea mays 1 cgcacgatag ccgccgccgt cgaccagagc actcccccgtcgtcgccacg atgtcgtctg 60 agcctccacc cgcctcctct gccgccgccg gagaggaactcgctgctgac ctttccgccg 120 ctaccctcag caagaagcag cagaagaagg acgcgaggaaggcggagaag gcagagcagc 180 gccagcgtca gcagcagcag cagcagcagc cggcggacgccgaggacccg ttcgcggcca 240 actacggcga ggtccccgtc gaggagatcc agtcaaaggccatctccggc cgctcgtggt 300 cccatgtcgg cgacctcgac gactccgctg cgggccgctccgtgcttatc cgcggagccg 360 cgcaggccat ccgtccggtc agcaagaaga tggctttcgtcgtgctgcgc cagagtatg 420 gcaccgtgca gtgcgtgctc gtcgccagcg ccgacgccggcgtcagcacg cagatggtc 480 gcttcgccac cgccctcagc aaggagtcca tcgtcgacgttgagggcgtc gtctccccc 540 caaaggagcc cctcaaggcc accacacagc aggttgagatccaagtgagg aagatcatt 600 gcatcaatag ggctattccg acccttccaa ttaaccttgaagatgcggct cggaggagg 660 cagattttga gaaggctgaa ttggctggag aaaagcttgttcgcgttggc caagtaccc 720 gcttgaacta cagagctatt gatctacgaa caccctcgaatcaagccata ttcggatcc 780 agtgtcaagt tgaaaacaaa tttagagatt ttttgttgtcgaagaacttt gtgggatcc 840 acaccccaaa attgatttct ggatctagtg aagggggtgcggctgtattc agcttctgt 900 acaatggtca acctgcttgt ttggcacaat cccctcagttatacaagcaa tggctatct 960 ctggtggttt tgagcgagta tttgaggtcg gccctgtgtttagagcagaa aattcaaaca 1020 cacacaggca tctatgtgag ttcgttggtc ttgatgctgaaatggagatt aaggagcatt 1080 attttgaggt ctgtgacatt atagatggct tattcgtatcaatatttaaa cacttgtctg 1140 aaaactgcaa gaaagaactc gaatcaataa acaggcagtatccatttgaa cctctgaagt 1200 atctagacaa aacctttaag ctcacttatg aagaaggaattcaaatgttg aaggaagccg 1260 gaacagaaat cgagcctatg ggtgacctca ataccgaagctgagaaaaaa cttggtcggc 1320 ttgtcaggga aaagtatgac acagattttt tcatcctgtatcggtatcct ttggctgtac 1380 gtccgttcta caccatgcct tgttatgaca acccagcgtacaccaattct tttgatgtct 1440 tcattcgagg cgaggagata atatctggag cacaaaggatacacactcct gagctgctgg 1500 ccaagcgcgc gacagagtgt ggaatcgacg tgagcactatctcggcctac attgaatcct 1560 tcagctatgg cgtgccgcca cacggcggtt tcggggtgggtttggagagg gtggtgatgc 1620 tgttctgtgc cctgaacaac atcaggaaga cctccctgttcccgcgcgac ccgcagaggc 1680 tcgtgccgta agtttctgat tccaagcctg agtcttcgagtggtctacgg agcagatccg 1740 atgttgttac catcagagtt gacttgcaat cttagctcctgaacctggcg gttaccgtgg 1800 atcagagttc ctgttgaatt tcacaaaagc ctacttgttcctaatagatt gctgcaacca 1860 acaatattac gaccctttcg ggcttttctt cccgcctcacgtgttattct ggtctatact 1920 tgtttttaag tgcaagtatt gctcagtt 1948 2 546 PRTZea mays 2 Met Ser Ser Glu Pro Pro Pro Ala Ser Ser Ala Ala Ala Gly GluGlu 1 5 10 15 Leu Ala Ala Asp Leu Ser Ala Ala Thr Leu Ser Lys Lys GlnGln Lys 20 25 30 Lys Asp Ala Arg Lys Ala Glu Lys Ala Glu Gln Arg Gln ArgGln Gln 35 40 45 Gln Gln Gln Gln Gln Pro Ala Asp Ala Glu Asp Pro Phe AlaAla Asn 50 55 60 Tyr Gly Glu Val Pro Val Glu Glu Ile Gln Ser Lys Ala IleSer Gly 65 70 75 80 Arg Ser Trp Ser His Val Gly Asp Leu Asp Asp Ser AlaAla Gly Arg 85 90 95 Ser Val Leu Ile Arg Gly Ala Ala Gln Ala Ile Arg ProVal Ser Lys 100 105 110 Lys Met Ala Phe Val Val Leu Arg Gln Ser Met SerThr Val Gln Cys 115 120 125 Val Leu Val Ala Ser Ala Asp Ala Gly Val SerThr Gln Met Val Arg 130 135 140 Phe Ala Thr Ala Leu Ser Lys Glu Ser IleVal Asp Val Glu Gly Val 145 150 155 160 Val Ser Leu Pro Lys Glu Pro LeuLys Ala Thr Thr Gln Gln Val Glu 165 170 175 Ile Gln Val Arg Lys Ile TyrCys Ile Asn Arg Ala Ile Pro Thr Leu 180 185 190 Pro Ile Asn Leu Glu AspAla Ala Arg Ser Glu Ala Asp Phe Glu Lys 195 200 205 Ala Glu Leu Ala GlyGlu Lys Leu Val Arg Val Gly Gln Asp Thr Arg 210 215 220 Leu Asn Tyr ArgAla Ile Asp Leu Arg Thr Pro Ser Asn Gln Ala Ile 225 230 235 240 Phe ArgIle Gln Cys Gln Val Glu Asn Lys Phe Arg Asp Phe Leu Leu 245 250 255 SerLys Asn Phe Val Gly Ile His Thr Pro Lys Leu Ile Ser Gly Ser 260 265 270Ser Glu Gly Gly Ala Ala Val Phe Lys Leu Leu Tyr Asn Gly Gln Pro 275 280285 Ala Cys Leu Ala Gln Ser Pro Gln Leu Tyr Lys Gln Met Ala Ile Ser 290295 300 Gly Gly Phe Glu Arg Val Phe Glu Val Gly Pro Val Phe Arg Ala Glu305 310 315 320 Asn Ser Asn Thr His Arg His Leu Cys Glu Phe Val Gly LeuAsp Ala 325 330 335 Glu Met Glu Ile Lys Glu His Tyr Phe Glu Val Cys AspIle Ile Asp 340 345 350 Gly Leu Phe Val Ser Ile Phe Lys His Leu Ser GluAsn Cys Lys Lys 355 360 365 Glu Leu Glu Ser Ile Asn Arg Gln Tyr Pro PheGlu Pro Leu Lys Tyr 370 375 380 Leu Asp Lys Thr Phe Lys Leu Thr Tyr GluGlu Gly Ile Gln Met Leu 385 390 395 400 Lys Glu Ala Gly Thr Glu Ile GluPro Met Gly Asp Leu Asn Thr Glu 405 410 415 Ala Glu Lys Lys Leu Gly ArgLeu Val Arg Glu Lys Tyr Asp Thr Asp 420 425 430 Phe Phe Ile Leu Tyr ArgTyr Pro Leu Ala Val Arg Pro Phe Tyr Thr 435 440 445 Met Pro Cys Tyr AspAsn Pro Ala Tyr Thr Asn Ser Phe Asp Val Phe 450 455 460 Ile Arg Gly GluGlu Ile Ile Ser Gly Ala Gln Arg Ile His Thr Pro 465 470 475 480 Glu LeuLeu Ala Lys Arg Ala Thr Glu Cys Gly Ile Asp Val Ser Thr 485 490 495 IleSer Ala Tyr Ile Glu Ser Phe Ser Tyr Gly Val Pro Pro His Gly 500 505 510Gly Phe Gly Val Gly Leu Glu Arg Val Val Met Leu Phe Cys Ala Leu 515 520525 Asn Asn Ile Arg Lys Thr Ser Leu Phe Pro Arg Asp Pro Gln Arg Leu 530535 540 Val Pro 545 3 730 DNA Oryza sativa 3 gcacgagctt acacggcacgagcttacagg aattcaaatg ctgaaggaag ctggaacaga 60 aatcgaaccc atgggtgacctcaacactga agctgagaaa aaactaggcc ggcttgttaa 120 ggagaagtat ggaacagaatttttcatcct ctatcggtat cctttggctg tgcgtccctt 180 ctacaccatg ccttgttatgacaacccagc ttacagtaac tcttttgatg tctttattcg 240 aggagaggaa ataatatctggagcacaaag aatacattta ccagagctat tgacgaaacg 300 tgcaacagag tgtggaattgatgcgagtac tatttcatca tatatcgaat cgttcagcta 360 tggtgcacct cctcatggtggttttggtgt cggcctggag agggtggtaa tgctgttctg 420 cgccctaaac aacatcaggaagacatcact tttccctcgc gatccacaaa ggctggtgcc 480 ataatttgct ttttttcccaagagcaaggt ttggactcag tacggactgg gcagttttcc 540 tcggctggtt tttttacctggacattattt tcgtatttat taatgtgctg tactgcaaaa 600 gctgctcctt tccacaacatttggaatagt tgccgataca tttggaatag ggctcaacgt 660 tggcgttgtg atttcgttgatgatcccgct attcgtaaca aaaaaaaaaa aaaaaaaaaa 720 aaaaaaaaaa 730 4 148 PRTOryza sativa 4 Met Leu Lys Glu Ala Gly Thr Glu Ile Glu Pro Met Gly AspLeu Asn 1 5 10 15 Thr Glu Ala Glu Lys Lys Leu Gly Arg Leu Val Lys GluLys Tyr Gly 20 25 30 Thr Glu Phe Phe Ile Leu Tyr Arg Tyr Pro Leu Ala ValArg Pro Phe 35 40 45 Tyr Thr Met Pro Cys Tyr Asp Asn Pro Ala Tyr Ser AsnSer Phe Asp 50 55 60 Val Phe Ile Arg Gly Glu Glu Ile Ile Ser Gly Ala GlnArg Ile His 65 70 75 80 Leu Pro Glu Leu Leu Thr Lys Arg Ala Thr Glu CysGly Ile Asp Ala 85 90 95 Ser Thr Ile Ser Ser Tyr Ile Glu Ser Phe Ser TyrGly Ala Pro Pro 100 105 110 His Gly Gly Phe Gly Val Gly Leu Glu Arg ValVal Met Leu Phe Cys 115 120 125 Ala Leu Asn Asn Ile Arg Lys Thr Ser LeuPhe Pro Arg Asp Pro Gln 130 135 140 Arg Leu Val Pro 145 5 1109 DNAGlycine max 5 gcacgaggtc atcagagaga atggcttcac cgttcaatgc ttggtgcaggcgcaggccga 60 tacggtgagc ccgcagatgg tgaagttcgc cgctgcactc agccgcgagtccatcgtcga 120 tgtcgaaggc gttgtttcga tcccctccgc tcccatcaaa ggcgccacacaacaggtgga 180 aattcaagtg aggaagttgt attgtgtcag tagggctgta cctactctgcctattaatct 240 tgaggatgct gctcgaagtg aagttgaaat cgagacggct cttcaggctggtgagcaact 300 tgttcgtgtt aatcaggata cacgtctgaa ctttagggtg cttgatgtgcgaacgccagc 360 taatcaaggg attttccgca ttcagtctca agttggaaat gcgtttagacaattcttatt 420 atctgaaggt ttttgtgaaa tccacactcc aaagttgata gctggatctagtgagggagg 480 agctgctgtt tttagactgg actacaaagg tcaacctgca tgcctggcccagtcacctca 540 gcttcacaag caaatgtcta tttgtggaga ttttggccgt gtttttgagattggtcctgt 600 gtttagagca gaagattcct acactcacag gcatctgtgt gagtttacaggtcttgatgt 660 tgaaatggag attaagaagc attactttga ggttatggat atagtcgatagattgtttgt 720 cgcaatgttt gacagtttga accagaattg taagaaggat ctggaagctgtcgggtctca 780 gtatccattt gaacctttga agtatctgcg gacgacacta cggcttacatatgaagaagg 840 gattcagatg ctcaaggatg ttggagtaga aattgaacct tatggtgacttgaatactga 900 agcggaaagg aaattgggtc agctagtctc agagaaatat ggcacagagttctatatcct 960 tcaccggtac cctttggctg taaggccatt ctatacaatg ccttgctacgacaatcctgc 1020 atacagcaac tcgtttgatg tctttattcg aggtgaggag ataatttcaggagctcagcg 1080 tgttcatgtg ccagaatttt tggaacaag 1109 6 369 PRT Glycinemax 6 His Glu Val Ile Arg Glu Asn Gly Phe Thr Val Gln Cys Leu Val Gln 15 10 15 Ala Gln Ala Asp Thr Val Ser Pro Gln Met Val Lys Phe Ala Ala Ala20 25 30 Leu Ser Arg Glu Ser Ile Val Asp Val Glu Gly Val Val Ser Ile Pro35 40 45 Ser Ala Pro Ile Lys Gly Ala Thr Gln Gln Val Glu Ile Gln Val Arg50 55 60 Lys Leu Tyr Cys Val Ser Arg Ala Val Pro Thr Leu Pro Ile Asn Leu65 70 75 80 Glu Asp Ala Ala Arg Ser Glu Val Glu Ile Glu Thr Ala Leu GlnAla 85 90 95 Gly Glu Gln Leu Val Arg Val Asn Gln Asp Thr Arg Leu Asn PheArg 100 105 110 Val Leu Asp Val Arg Thr Pro Ala Asn Gln Gly Ile Phe ArgIle Gln 115 120 125 Ser Gln Val Gly Asn Ala Phe Arg Gln Phe Leu Leu SerGlu Gly Phe 130 135 140 Cys Glu Ile His Thr Pro Lys Leu Ile Ala Gly SerSer Glu Gly Gly 145 150 155 160 Ala Ala Val Phe Arg Leu Asp Tyr Lys GlyGln Pro Ala Cys Leu Ala 165 170 175 Gln Ser Pro Gln Leu His Lys Gln MetSer Ile Cys Gly Asp Phe Gly 180 185 190 Arg Val Phe Glu Ile Gly Pro ValPhe Arg Ala Glu Asp Ser Tyr Thr 195 200 205 His Arg His Leu Cys Glu PheThr Gly Leu Asp Val Glu Met Glu Ile 210 215 220 Lys Lys His Tyr Phe GluVal Met Asp Ile Val Asp Arg Leu Phe Val 225 230 235 240 Ala Met Phe AspSer Leu Asn Gln Asn Cys Lys Lys Asp Leu Glu Ala 245 250 255 Val Gly SerGln Tyr Pro Phe Glu Pro Leu Lys Tyr Leu Arg Thr Thr 260 265 270 Leu ArgLeu Thr Tyr Glu Glu Gly Ile Gln Met Leu Lys Asp Val Gly 275 280 285 ValGlu Ile Glu Pro Tyr Gly Asp Leu Asn Thr Glu Ala Glu Arg Lys 290 295 300Leu Gly Gln Leu Val Ser Glu Lys Tyr Gly Thr Glu Phe Tyr Ile Leu 305 310315 320 His Arg Tyr Pro Leu Ala Val Arg Pro Phe Tyr Thr Met Pro Cys Tyr325 330 335 Asp Asn Pro Ala Tyr Ser Asn Ser Phe Asp Val Phe Ile Arg GlyGlu 340 345 350 Glu Ile Ile Ser Gly Ala Gln Arg Val His Val Pro Glu PheLeu Glu 355 360 365 Gln 7 836 DNA Triticum aestivum 7 tacacatgcagactttcagt gagtttttgt tctcggactt gggatccaca gtccaaagtt 60 gattggtggatcaagtgaac ttggtgcatc tccattcaag ctggcgtaca attaccaacc 120 tgcttatttagcgcagtctc tacaatcata caagcaaatg agcatctgtg gtggctttgg 180 gcgcgtgtttgaggctggtc cggtatttag atcagaaaaa tcaaacactc acaggcatct 240 atgtgagtttattgggttgg atgcagaaat ggagattaag gagcactact ttgaggtttg 300 tgatatcatagattgctaat tgtagcaata ttcaaacacc caaatgaaaa ttgtcagaag 360 gaactcgagacaataaatag gcagtatcca tttgaacctc tgaagtacct agagaaaacg 420 ttgaagctaacgtacgagga agggattaaa atgctcaagg tttcattctg gaatcctcta 480 ggcagggtgcttgcaatccc ctacatctcg gctgcaacaa aaaagaccca acgaggctgt 540 tgtttcaagctcagaccctc ttcattgcac gcggtgctag aaggagaact gggttgtggt 600 gctgttgctggtcgttttcc tttttacttt tgcactttgg ccgtcataaa cgatacatgc 660 ttgctccctggatggatctc tttctctccc tggatctttt aaacaggtgt tgtgattaaa 720 attgtgataaatcagtgttc atcactaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780 aatctcgagggggggcccgg tactgttcac cgcgtggcgc cgggctagag actagt 836 8 98 PRT Triticumaestivum 8 Val Phe Val Leu Gly Leu Gly Ile His Ser Pro Lys Leu Ile GlyGly 1 5 10 15 Ser Ser Glu Leu Gly Ala Ser Pro Phe Lys Leu Ala Tyr AsnTyr Gln 20 25 30 Pro Ala Tyr Leu Ala Gln Ser Leu Gln Ser Tyr Lys Gln MetSer Ile 35 40 45 Cys Gly Gly Phe Gly Arg Val Phe Glu Ala Gly Pro Val PheArg Ser 50 55 60 Glu Lys Ser Asn Thr His Arg His Leu Cys Glu Phe Ile GlyLeu Asp 65 70 75 80 Ala Glu Met Glu Ile Lys Glu His Tyr Phe Glu Val CysAsp Ile Ile 85 90 95 Asp Cys 9 2085 DNA Zea mays 9 ggaaaccgtg tttcgacgggccgcagtggg cagtggcttg gcccatcgaa cccacttgcc 60 actcacttcc acctgaactttgccctgcct tctctcgacg actcccctgt ccccgccgcc 120 gccgccgccg caaatccccttccgcgtctg tctggcctct ggggcttcta ggttagcgcg 180 tgcgaccacc atggccgaggaggtccaggc tccactttcc gccaccatgg cgaaggaggc 240 ccagtcgccg ccgtccgcaaccatagcgga ggcgacggcg ccgccgcagc tcttattatt 300 taactccttt acgaagagggaggagccatt ccagccccgg gtagagggga aggtagggat 360 gtacgtctgt ggcgtcactccctacgactt tagccacatc ggccacgcgc gtgcctacgt 420 cgccttcgac gtcctctacaggtaccttaa attcttgggg tatgaagttg aatatgtccg 480 taatttcacg gatattgatgacaagattat taagcgtgcc aatgaacgcg gtgaaacagt 540 aacaagcttg agtagccagtttatcaatga atttcttctt gacatgactg agctccagtg 600 cttgcctcct acctgcgagccacgggtaac agaacacatt gagcatatta taaagttgat 660 aacacagata atggagaatggcaaagccta tgctattgaa ggagatgttt acttttcagt 720 tgaaagtttt cctgaatatctcagtttatc tggaagaaaa tttgatcaaa atcaggcagg 780 tgcacgggtt gcttttgatacaagaaagcg taatcctgca gacttcgcac tctggaaagc 840 tgcaaaggag ggtgaacctttttgggatag cccttggggc cgtggaagac caggttggca 900 tattgaatgc agcgcaatgagtgctcacta tttaggacat gtattcgata ttcatggtgg 960 ggggaaagat ttgatttttcctcatcatga gaatgagctt gcacaaagcc gcgcagctta 1020 tcctgatagc gaggtcaaatgctggatgca caatggcttt gttaacaagg atgataaaaa 1080 aatggcaaaa tcagataataactttttcac gattagagat atcattgctc tttaccatcc 1140 aatggcttta agatttttcttgatgcgcac acattataga tcagatgtta accattctga 1200 tcaagcgctt gagattgcatctgatcgtgt ctactacatt tatcagactc tatatgactg 1260 tgaggaagtg ttagctacatatcgtgaaga gggtacctct ctcccagtgc cgtctgagga 1320 gcaaaatctg attggtaagcaccattcaga attcttgaaa catatgtcga atgatcttaa 1380 aaccacagat gttctggaccgttgcttcat ggagctgctg aaggccataa acagcagtct 1440 gaatgatttg aagaaactgcagcaaaaaat agaacagcaa aagaagaaac agcaacagca 1500 gaagaagcag caacagcagaagcagcagca acagaagcaa cagcaattgc aaaaacagcc 1560 agaagattat attcaagctctgattgcact ggaaacagaa cttaaaaaca aattgtctat 1620 acttggtctg atgccatcttcatctttggc agaggtactg aagcaattga aggacaaatc 1680 attaaagcga gcagggctgactgaagaaca attgcaagag cagattgagc agagaaatgt 1740 cgcaaggaag aataagcagtttgagatatc tgatggaatc aggaaaaacc ttgctaccaa 1800 aggcatcgcc ctgatggacgaaccttctgg tacagtatgg agaccatgcg aaccagagcg 1860 gtctgaagag tcatgattagctcactgact caacaagtga tggcggtgta aaatgagatt 1920 tttgcctgag ggcagttatcgcattttgaa gactaacaaa aatcgccatc tctggatgtg 1980 gtattctaca gggtaggggttccaggttga ctcaccagtt aaaacatgca tttctggttg 2040 tataacaagc aatgaaccccatatatatac ttgacagttg actcc 2085 10 599 PRT Zea mays 10 Thr Leu Pro CysLeu Leu Ser Thr Thr Pro Leu Ser Pro Pro Pro Pro 1 5 10 15 Pro Pro GlnIle Pro Phe Arg Val Cys Leu Ala Ser Gly Ala Ser Arg 20 25 30 Leu Ala ArgAla Thr Thr Met Ala Glu Glu Val Gln Ala Pro Leu Ser 35 40 45 Ala Thr MetAla Lys Glu Ala Gln Ser Pro Pro Ser Ala Thr Ile Ala 50 55 60 Glu Ala ThrAla Pro Pro Gln Leu Leu Leu Phe Asn Ser Phe Thr Lys 65 70 75 80 Arg GluGlu Pro Phe Gln Pro Arg Val Glu Gly Lys Val Gly Met Tyr 85 90 95 Val CysGly Val Thr Pro Tyr Asp Phe Ser His Ile Gly His Ala Arg 100 105 110 AlaTyr Val Ala Phe Asp Val Leu Tyr Arg Tyr Leu Lys Phe Leu Gly 115 120 125Tyr Glu Val Glu Tyr Val Arg Asn Phe Thr Asp Ile Asp Asp Lys Ile 130 135140 Ile Lys Arg Ala Asn Glu Arg Gly Glu Thr Val Thr Ser Leu Ser Ser 145150 155 160 Gln Phe Ile Asn Glu Phe Leu Leu Asp Met Thr Glu Leu Gln CysLeu 165 170 175 Pro Pro Thr Cys Glu Pro Arg Val Thr Glu His Ile Glu HisIle Ile 180 185 190 Lys Leu Ile Thr Gln Ile Met Glu Asn Gly Lys Ala TyrAla Ile Glu 195 200 205 Gly Asp Val Tyr Phe Ser Val Glu Ser Phe Pro GluTyr Leu Ser Leu 210 215 220 Ser Gly Arg Lys Phe Asp Gln Asn Gln Ala GlyAla Arg Val Ala Phe 225 230 235 240 Asp Thr Arg Lys Arg Asn Pro Ala AspPhe Ala Leu Trp Lys Ala Ala 245 250 255 Lys Glu Gly Glu Pro Phe Trp AspSer Pro Trp Gly Arg Gly Arg Pro 260 265 270 Gly Trp His Ile Glu Cys SerAla Met Ser Ala His Tyr Leu Gly His 275 280 285 Val Phe Asp Ile His GlyGly Gly Lys Asp Leu Ile Phe Pro His His 290 295 300 Glu Asn Glu Leu AlaGln Ser Arg Ala Ala Tyr Pro Asp Ser Glu Val 305 310 315 320 Lys Cys TrpMet His Asn Gly Phe Val Asn Lys Asp Asp Lys Lys Met 325 330 335 Ala LysSer Asp Asn Asn Phe Phe Thr Ile Arg Asp Ile Ile Ala Leu 340 345 350 TyrHis Pro Met Ala Leu Arg Phe Phe Leu Met Arg Thr His Tyr Arg 355 360 365Ser Asp Val Asn His Ser Asp Gln Ala Leu Glu Ile Ala Ser Asp Arg 370 375380 Val Tyr Tyr Ile Tyr Gln Thr Leu Tyr Asp Cys Glu Glu Val Leu Ala 385390 395 400 Thr Tyr Arg Glu Glu Gly Thr Ser Leu Pro Val Pro Ser Glu GluGln 405 410 415 Asn Leu Ile Gly Lys His His Ser Glu Phe Leu Lys His MetSer Asn 420 425 430 Asp Leu Lys Thr Thr Asp Val Leu Asp Arg Cys Phe MetGlu Leu Leu 435 440 445 Lys Ala Ile Asn Ser Ser Leu Asn Asp Leu Lys LysLeu Gln Gln Lys 450 455 460 Ile Glu Gln Gln Lys Lys Lys Gln Gln Gln GlnLys Lys Gln Gln Gln 465 470 475 480 Gln Lys Gln Gln Gln Gln Lys Gln GlnGln Leu Gln Lys Gln Pro Glu 485 490 495 Asp Tyr Ile Gln Ala Leu Ile AlaLeu Glu Thr Glu Leu Lys Asn Lys 500 505 510 Leu Ser Ile Leu Gly Leu MetPro Ser Ser Ser Leu Ala Glu Val Leu 515 520 525 Lys Gln Leu Lys Asp LysSer Leu Lys Arg Ala Gly Leu Thr Glu Glu 530 535 540 Gln Leu Gln Glu GlnIle Glu Gln Arg Asn Val Ala Arg Lys Asn Lys 545 550 555 560 Gln Phe GluIle Ser Asp Gly Ile Arg Lys Asn Leu Ala Thr Lys Gly 565 570 575 Ile AlaLeu Met Asp Glu Pro Ser Gly Thr Val Trp Arg Pro Cys Glu 580 585 590 ProGlu Arg Ser Glu Glu Ser 595 11 1957 DNA Oryza sativa 11 cgccagttctagggttagct cgtcggcgtc cagccctctc actctccccc tccgctctca 60 cgatggcggagagcgcgaag ccgacgccgc agctggagct cttcaactcg atgacgaaga 120 agaaggagctcttcgagccg cttgtggagg ggaaggtccg catgtatgtg tgcggcgtca 180 cgccctacgacttcagccac atcggccacg cccgcgccta cgtcgccttc gacgtcctct 240 acaggtatcttaaattcttg gggtacgagg tcgaatatgt gcgcaacttc actgatattg 300 atgacaagattatcaaacga gcaaatgaag ctggtgaaac tgtaactagc ttgagcagcc 360 ggtttattaatgaattcctt ctcgatatgg ctcagctcca gtgcttaccc ccaacttgtg 420 agccacgtgtgacggatcac attgaacata ttatagagtt gataaccaag ataatggaga 480 atgggaaagcctatgctatg gaaggagatg tttacttttc agttgatact ttccctgagt 540 atctcagtttatctggaagg aagttagatc ataatcttgc tggttcgcgg gttgctgtcg 600 atacaagaaagcggaaccct gcagactttg cgctgtggaa ggctgctaag gaaggcgaac 660 ctttctgggatagcccatgg ggccgtggta gaccaggatg gcatattgaa tgcagtgcaa 720 tgagtgctcattatttagga catgtgtttg atatccatgg tggagggaaa gatctgatat 780 ttcctcatcatgagaatgag cttgctcaga gccgggcagc ttatccagaa agtgaggtca 840 aatgttggatgcacaatggg tttgttaaca aggatgatca gaaaatgtca aagtcagata 900 aaaatttcttcacaatccga gatattattg atctgtacca tcccatggct ttgaggtttt 960 tcctgatgcgcacacattac agaggagatg tgaatcactc tgacaaagca cttgagatag 1020 catctgatcgtgtctactac atatatcaga ctttatatga ctgtgaggaa gtgttgtctc 1080 aatatcgtggagagaatatc tctgtcccgg tccctgttga ggaacaagat atggttaaca 1140 agcaccattcagaattcttg gaatctatgg cggatgatct tagaacaaca gatgttctgg 1200 atggctttactgacttgctg aaggcaatta acagcaattt gaatgatttt aagaagttgc 1260 aacagaagctagagcagcaa aagaagaaac aacaacagca gaagcagcag aagcaaaagc 1320 agcagcaggcacagaaacaa ccagaagaat atattcaagc tatgtttgca cttgagacag 1380 aaattaaaaataaaatatct atccttggtc tgatgccacc ttcttccttg gcagaggcac 1440 tgaagcaacttaaggataaa gctttgaaga gagcagggtt gactgaagaa ctgttgcagg 1500 agcaaattgagcagagaact gctgcaagga aaaacaagca gtttgatgtg tctgaccaaa 1560 tcaggaaacagctaggcagc aaaggcatag ccctcatgga tgaacctact ggtacagtat 1620 ggagaccatgcgagccagag tctgaatagt cacatgattg atttgtgctt tggttaacag 1680 gtgatggtacaaactggaaa atttaaccaa gcacatctgc tgaattggtg taaattgatg 1740 cagatcaacatttttttttg taattttgta ggggtttaag ttcactggcc aactgaaact 1800 tgcgtttctcgtggtgtaag aagcaaaacc ccatatactg atatactcga ggactccctt 1860 gttggatgttatgctttgga tttgaatatt gaagtcaaat cataattaca tttgcatgat 1920 caaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaa 1957 12 548 PRT Oryza sativa 12 Pro ValLeu Gly Leu Ala Arg Arg Arg Pro Ala Leu Ser Leu Ser Pro 1 5 10 15 SerAla Leu Thr Met Ala Glu Ser Ala Lys Pro Thr Pro Gln Leu Glu 20 25 30 LeuPhe Asn Ser Met Thr Lys Lys Lys Glu Leu Phe Glu Pro Leu Val 35 40 45 GluGly Lys Val Arg Met Tyr Val Cys Gly Val Thr Pro Tyr Asp Phe 50 55 60 SerHis Ile Gly His Ala Arg Ala Tyr Val Ala Phe Asp Val Leu Tyr 65 70 75 80Arg Tyr Leu Lys Phe Leu Gly Tyr Glu Val Glu Tyr Val Arg Asn Phe 85 90 95Thr Asp Ile Asp Asp Lys Ile Ile Lys Arg Ala Asn Glu Ala Gly Glu 100 105110 Thr Val Thr Ser Leu Ser Ser Arg Phe Ile Asn Glu Phe Leu Leu Asp 115120 125 Met Ala Gln Leu Gln Cys Leu Pro Pro Thr Cys Glu Pro Arg Val Thr130 135 140 Asp His Ile Glu His Ile Ile Glu Leu Ile Thr Lys Ile Met GluAsn 145 150 155 160 Gly Lys Ala Tyr Ala Met Glu Gly Asp Val Tyr Phe SerVal Asp Thr 165 170 175 Phe Pro Glu Tyr Leu Ser Leu Ser Gly Arg Lys LeuAsp His Asn Leu 180 185 190 Ala Gly Ser Arg Val Ala Val Asp Thr Arg LysArg Asn Pro Ala Asp 195 200 205 Phe Ala Leu Trp Lys Ala Ala Lys Glu GlyGlu Pro Phe Trp Asp Ser 210 215 220 Pro Trp Gly Arg Gly Arg Pro Gly TrpHis Ile Glu Cys Ser Ala Met 225 230 235 240 Ser Ala His Tyr Leu Gly HisVal Phe Asp Ile His Gly Gly Gly Lys 245 250 255 Asp Leu Ile Phe Pro HisHis Glu Asn Glu Leu Ala Gln Ser Arg Ala 260 265 270 Ala Tyr Pro Glu SerGlu Val Lys Cys Trp Met His Asn Gly Phe Val 275 280 285 Asn Lys Asp AspGln Lys Met Ser Lys Ser Asp Lys Asn Phe Phe Thr 290 295 300 Ile Arg AspIle Ile Asp Leu Tyr His Pro Met Ala Leu Arg Phe Phe 305 310 315 320 LeuMet Arg Thr His Tyr Arg Gly Asp Val Asn His Ser Asp Lys Ala 325 330 335Leu Glu Ile Ala Ser Asp Arg Val Tyr Tyr Ile Tyr Gln Thr Leu Tyr 340 345350 Asp Cys Glu Glu Val Leu Ser Gln Tyr Arg Gly Glu Asn Ile Ser Val 355360 365 Pro Val Pro Val Glu Glu Gln Asp Met Val Asn Lys His His Ser Glu370 375 380 Phe Leu Glu Ser Met Ala Asp Asp Leu Arg Thr Thr Asp Val LeuAsp 385 390 395 400 Gly Phe Thr Asp Leu Leu Lys Ala Ile Asn Ser Asn LeuAsn Asp Phe 405 410 415 Lys Lys Leu Gln Gln Lys Leu Glu Gln Gln Lys LysLys Gln Gln Gln 420 425 430 Gln Lys Gln Gln Lys Gln Lys Gln Gln Gln AlaGln Lys Gln Pro Glu 435 440 445 Glu Tyr Ile Gln Ala Met Phe Ala Leu GluThr Glu Ile Lys Asn Lys 450 455 460 Ile Ser Ile Leu Gly Leu Met Pro ProSer Ser Leu Ala Glu Ala Leu 465 470 475 480 Lys Gln Leu Lys Asp Lys AlaLeu Lys Arg Ala Gly Leu Thr Glu Glu 485 490 495 Leu Leu Gln Glu Gln IleGlu Gln Arg Thr Ala Ala Arg Lys Asn Lys 500 505 510 Gln Phe Asp Val SerAsp Gln Ile Arg Lys Gln Leu Gly Ser Lys Gly 515 520 525 Ile Ala Leu MetAsp Glu Pro Thr Gly Thr Val Trp Arg Pro Cys Glu 530 535 540 Pro Glu SerGlu 545 13 2183 DNA Glycine max 13 gcacgagata aacgataacg ttatttggctgtgaatttgg gatgagctgg tccggtgcaa 60 aaatgggtac ggtgtctctt ctcaagtgctacagaccctt tttctctatg cttttccctc 120 actccgctcc acccagactc cacgccgccatcttcaggag caaaaacttt tctttttgcg 180 ccacctcgtc cccgccgttg acggcggagaagggttgcgg caaatccgac gccgagtgtc 240 ccaccttgcc ggaggtgtgg ctgcacaacaccatgagtag gacgaaggaa ctcttcaaac 300 ccaaagtgga atccaaagtg ggaatgtacgtgtgcggcgt caccgcttat gatcttagcc 360 atattggaca cgctcgcgta tacgtcaatttcgaccttct ttacagatac tttaagcatt 420 tgggatttga agtctgttat gttcgcaatttcactgacgt agatgacaag ataattgcta 480 gagcaaagga gttaggagaa gatccaatcagtttgagctg gcgctattgt gaagagttct 540 gtcaagacat ggtaactctt aattgtctgtctccctctgt ggaaccaaag gtctcagagc 600 acatgcccca aatcattgat atgattgagaagatccttaa taatgggtat gcctacattg 660 ttgatgggga tgtgtacttt aatgtagaaaaatttccaga atatgggaaa ctatctagtc 720 gagatctaga agataatcga gctggtgagagggttgcagt tgattctaga aagaaaaatc 780 ctgctgattt tgctctttgg aagtctgcaaagccagggga gccattttgg gagagtccct 840 ggggtcctgg aagacctggg tggcatattgaatgcagtgc catgagtgca gcttatcttg 900 gttactcttt tgatatccat ggtggaggaatcgaccttgt gtttcctcac catgagaatg 960 aaattgctca gagttgtgct gcatgtaagaaaagtgatat aagtatatgg atgcacaatg 1020 gttttgtcac cattgactct gtgaaaatgtcaaaatcttt ggggaatttt ttcacaatac 1080 gtcaggttat agacgtttac catccactggccttgagata ttttttgatg agcgcacatt 1140 atcgatctcc tattaactac tcaaatatacagctcgaaag tgcttcagac cgtgtttttt 1200 atatatatga gacattacat gaatgtgaaagctttttgaa tcagcatgat cagaggaagg 1260 attccacccc accggatact ttggatattattgataagtt ccacgatgtt tttttgacct 1320 caatgtcgga tgatcttcac actccagttgtattggctgg aatgtctgat ccattaaaat 1380 caatcaatga tttgctgcat gctcgtaaggggaaaaaaca acaatttcga atcgaatcac 1440 tatcagcttt ggagaagagc gtcagggatgtccttactgt tttaggactt atgcctgcaa 1500 gttactctga ggttttgcag cagcttaaggtaaaagcttt aaaacgtgca aactttacgg 1560 aagaagaagt cttgcagaaa attgaagaacgggctactgc tagaatgcaa aaggagtatg 1620 ctaaatcgga tgcaatcagg aaggatttggctgtacttgg tattactctt atggacagtc 1680 caaatggcac aacttggagg cctgccattcctcttccact tcaagagctg ctctaagtca 1740 agagttgttc aacatctcca aagcaaaaccaagaaatgta agttactagg ttctggtata 1800 tggaaatcaa ttataaggga tgccacgggtgtatctcgct atcaacttct cagaatgata 1860 aaggcgaccc cttcttaact cttgatgccgtaaaaacatg gattacaatt tacgttttga 1920 tagagatgtg cttagtgtag ttgtcttggtgaccaatatt gaattttttt tttttcttca 1980 tataccgggc ttttaacccc tagagtattcatagtttcaa cgaatttgag tttcagatta 2040 atattaaaat aaatagtcgc actatcactagagtagtgtt atgtttctac tttctagagt 2100 agcttcggtt taatattgag aaagacattttttttgtggt gataatgaat tttctgttgt 2160 tttttaaaaa aaaaaaaaaa aaa 2183 14574 PRT Glycine max 14 Thr Ile Thr Leu Phe Gly Cys Glu Phe Gly Met SerTrp Ser Gly Ala 1 5 10 15 Lys Met Gly Thr Val Ser Leu Leu Lys Cys TyrArg Pro Phe Phe Ser 20 25 30 Met Leu Phe Pro His Ser Ala Pro Pro Arg LeuHis Ala Ala Ile Phe 35 40 45 Arg Ser Lys Asn Phe Ser Phe Cys Ala Thr SerSer Pro Pro Leu Thr 50 55 60 Ala Glu Lys Gly Cys Gly Lys Ser Asp Ala GluCys Pro Thr Leu Pro 65 70 75 80 Glu Val Trp Leu His Asn Thr Met Ser ArgThr Lys Glu Leu Phe Lys 85 90 95 Pro Lys Val Glu Ser Lys Val Gly Met TyrVal Cys Gly Val Thr Ala 100 105 110 Tyr Asp Leu Ser His Ile Gly His AlaArg Val Tyr Val Asn Phe Asp 115 120 125 Leu Leu Tyr Arg Tyr Phe Lys HisLeu Gly Phe Glu Val Cys Tyr Val 130 135 140 Arg Asn Phe Thr Asp Val AspAsp Lys Ile Ile Ala Arg Ala Lys Glu 145 150 155 160 Leu Gly Glu Asp ProIle Ser Leu Ser Trp Arg Tyr Cys Glu Glu Phe 165 170 175 Cys Gln Asp MetVal Thr Leu Asn Cys Leu Ser Pro Ser Val Glu Pro 180 185 190 Lys Val SerGlu His Met Pro Gln Ile Ile Asp Met Ile Glu Lys Ile 195 200 205 Leu AsnAsn Gly Tyr Ala Tyr Ile Val Asp Gly Asp Val Tyr Phe Asn 210 215 220 ValGlu Lys Phe Pro Glu Tyr Gly Lys Leu Ser Ser Arg Asp Leu Glu 225 230 235240 Asp Asn Arg Ala Gly Glu Arg Val Ala Val Asp Ser Arg Lys Lys Asn 245250 255 Pro Ala Asp Phe Ala Leu Trp Lys Ser Ala Lys Pro Gly Glu Pro Phe260 265 270 Trp Glu Ser Pro Trp Gly Pro Gly Arg Pro Gly Trp His Ile GluCys 275 280 285 Ser Ala Met Ser Ala Ala Tyr Leu Gly Tyr Ser Phe Asp IleHis Gly 290 295 300 Gly Gly Ile Asp Leu Val Phe Pro His His Glu Asn GluIle Ala Gln 305 310 315 320 Ser Cys Ala Ala Cys Lys Lys Ser Asp Ile SerIle Trp Met His Asn 325 330 335 Gly Phe Val Thr Ile Asp Ser Val Lys MetSer Lys Ser Leu Gly Asn 340 345 350 Phe Phe Thr Ile Arg Gln Val Ile AspVal Tyr His Pro Leu Ala Leu 355 360 365 Arg Tyr Phe Leu Met Ser Ala HisTyr Arg Ser Pro Ile Asn Tyr Ser 370 375 380 Asn Ile Gln Leu Glu Ser AlaSer Asp Arg Val Phe Tyr Ile Tyr Glu 385 390 395 400 Thr Leu His Glu CysGlu Ser Phe Leu Asn Gln His Asp Gln Arg Lys 405 410 415 Asp Ser Thr ProPro Asp Thr Leu Asp Ile Ile Asp Lys Phe His Asp 420 425 430 Val Phe LeuThr Ser Met Ser Asp Asp Leu His Thr Pro Val Val Leu 435 440 445 Ala GlyMet Ser Asp Pro Leu Lys Ser Ile Asn Asp Leu Leu His Ala 450 455 460 ArgLys Gly Lys Lys Gln Gln Phe Arg Ile Glu Ser Leu Ser Ala Leu 465 470 475480 Glu Lys Ser Val Arg Asp Val Leu Thr Val Leu Gly Leu Met Pro Ala 485490 495 Ser Tyr Ser Glu Val Leu Gln Gln Leu Lys Val Lys Ala Leu Lys Arg500 505 510 Ala Asn Phe Thr Glu Glu Glu Val Leu Gln Lys Ile Glu Glu ArgAla 515 520 525 Thr Ala Arg Met Gln Lys Glu Tyr Ala Lys Ser Asp Ala IleArg Lys 530 535 540 Asp Leu Ala Val Leu Gly Ile Thr Leu Met Asp Ser ProAsn Gly Thr 545 550 555 560 Thr Trp Arg Pro Ala Ile Pro Leu Pro Leu GlnGlu Leu Leu 565 570 15 633 DNA Zea mays 15 gcacacacgt cggtccaaacacgcgccgtc cgctcgcggc ttctccaacc aaagccgtgc 60 agccaaatcc gaagggtagcgtagcacggg gacgacgcca tgagccgcgc gctcctctcc 120 cacgtcctcc accgtccgccgcacttcgcg tacacctgct taaggagtgg cgttggtgcc 180 cgaggaggag tgctcgcttctggcatccac ccactccgtc gtctcaattg cagcgcggtt 240 gaagccgttc ccggccccaccgaggaggcg cctgctcctc aggcaaggaa gaaaagagta 300 gtttctggtg tacagccaacaggatcggtt caccttggaa attatctagg ggcaattaag 360 aattgggttg cacttcaggattcatatgag acattctttt tcatcgtgga tcttcatgca 420 attactttac catatgaggcgccactgctt tctaaagcaa caagaagcac tgctgcaata 480 tatcttgcat gtggcgtcgacagctccaag gcttctatct ttgtacagtc tcatgtccgt 540 gctcatgttg agttgatgtggctattgagt tcttctactc ctattggctg gctgaataga 600 atgatccagt tcaaagagaagtctcgcaag gcg 633 16 410 PRT Zea mays 16 His Gly Asp Asp Ala Met SerArg Ala Leu Leu Ser His Val Leu His 1 5 10 15 Arg Pro Pro His Phe AlaTyr Thr Cys Leu Arg Ser Gly Val Gly Ala 20 25 30 Arg Gly Gly Val Leu AlaSer Gly Ile His Pro Leu Arg Arg Leu Asn 35 40 45 Cys Ser Ala Val Glu AlaVal Pro Gly Pro Thr Glu Glu Ala Pro Ala 50 55 60 Pro Gln Ala Arg Lys LysArg Val Val Ser Gly Val Gln Pro Thr Gly 65 70 75 80 Ser Val His Leu GlyAsn Tyr Leu Gly Ala Ile Lys Asn Trp Val Ala 85 90 95 Leu Gln Asp Ser TyrGlu Thr Phe Phe Phe Ile Val Asp Leu His Ala 100 105 110 Ile Thr Leu ProTyr Glu Ala Pro Leu Leu Ser Lys Ala Thr Arg Ser 115 120 125 Thr Ala AlaIle Tyr Leu Ala Cys Gly Val Asp Ser Ser Lys Ala Ser 130 135 140 Ile PheVal Gln Ser His Val Arg Ala His Val Glu Leu Met Trp Leu 145 150 155 160Leu Ser Ser Ser Thr Pro Ile Gly Trp Leu Asn Arg Met Ile Gln Phe 165 170175 Lys Glu Lys Ser Arg Lys Ala Gly Asp Glu Asn Val Gly Val Ala Leu 180185 190 Leu Thr Tyr Pro Val Leu Met Ala Ser Asp Ile Leu Leu Tyr Gln Ser195 200 205 Asp Leu Val Pro Val Gly Glu Asp Gln Thr Gln His Leu Glu LeuThr 210 215 220 Arg Glu Ile Ala Glu Arg Val Asn Asn Leu Tyr Gly Gly ArgLys Trp 225 230 235 240 Lys Lys Leu Gly Gly Arg Gly Gly Leu Leu Phe LysVal Pro Glu Ala 245 250 255 Leu Ile Pro Pro Ala Gly Ala Arg Val Met SerLeu Thr Asp Gly Leu 260 265 270 Ser Lys Met Ser Lys Ser Ala Pro Ser AspGln Ser Arg Ile Asn Leu 275 280 285 Leu Asp Pro Lys Asp Val Ile Ala AsnLys Ile Lys Arg Cys Lys Thr 290 295 300 Asp Ser Phe Pro Gly Met Glu PheAsp Asn Pro Glu Arg Pro Glu Cys 305 310 315 320 Arg Asn Leu Leu Ser IleTyr Gln Ile Ile Thr Glu Lys Thr Lys Glu 325 330 335 Glu Val Val Ser GluCys Gln His Met Asn Trp Gly Thr Phe Lys Thr 340 345 350 Thr Leu Thr GluAla Leu Ile Asp His Leu Gln Pro Ile Gln Val Arg 355 360 365 Tyr Glu GluIle Met Ser Asp Pro Ala Tyr Leu Asp Asn Val Leu Leu 370 375 380 Glu GlyAla Val Lys Ala Ala Glu Ile Ala Asp Ile Thr Leu Asn Asn 385 390 395 400Val Tyr Gln Ala Met Gly Phe Leu Arg Arg 405 410 17 1536 DNA Glycine max17 gcacgaggga agatgagcgt ttcacatttc gcggttctat cgtcgtgttg ttgtccacgc 60ttggcccctt ctctgtcgcg tgcttcaacc cttcgttctc gcatccggtg ttgtactact 120ctcactgcta cttcttcaga gactcccact ccaaccttcg tgaagaaacg agtagtgtcg 180ggggttcagc ccacgggctc aattcacctc ggaaactatt ttggcgccat caagaattgg 240gttgcccttc agaatgtgta tgatacactt ttcttcattg tggacctgca cgcgattaca 300ttaccatatg acacccaaca attatctaag gctacaaggt caactgctgc tatttaccta 360gcatgtggag tggatccttc aaaggcttca gtatttgtac agtctcatgt tcgggcacat 420gtagaattga tgtggctgct aagttccaca acaccaattg gttggctgaa caaaatgata 480caatttaaag agaaatctcg caaggcggga gatgaagaag ttggggttgc ccttttgact 540tatcctgttc tgatggcttc tgatatactt ctatatcagt ctgattttgt ccctgttggt 600gaagatcaaa agcagcactt ggagttgact cgtgacttgg ctgaacgggt taataattta 660tatggaggaa gaaagtggaa gaaattaggc ggttatgaca gccgaggtgg tactatattt 720aaggttccag agccccttat acctccagcc ggagcccgga taatgtccct aactgatggc 780ctgtccaaga tgtcaaagtc tgcaccttct gatcaatcca gaatcaatat tcttgatcct 840aaagatctca tagcaaacaa gatcaaacgt tgcaaaactg attcatttcc tggcttggaa 900tttgacaact ctgagaggcc tgaatgtaac aatcttgttt ccatatacca gcttatttca 960ggaaagacga aagaggaagt tgtgcaggaa tgccaaaaca tgaactgggg cacattcaaa 1020cctcttttaa cagatgcctt gattgatcat ttgcatccca ttcaggttcg ctatgaggaa 1080atcatgtccg attcaggtta tttagatgga gttttagcac aaggtgctag aaatgcagca 1140gatatagcag attctacact taataatatt taccaagcaa tgggattttt taagagacag 1200tgataattga tgccaaataa attaaagatt ggcgagacgt caacttaaaa gctaacttct 1260ggatgattca tgatgggcct caaaattttg gagtaatctt atggacatat acttgactac 1320tggaaatgga aagattattg atgcaaagcc taaaggtccc attagttctt gatgcaatgg 1380gctttgtatc tccttcattt ttctccgagt atggtcgttg ccttcatttt atattttatt 1440gtttcaatct ctttcattat ttacttgtat tttataatga attcagcata ttgataaatt 1500gttccgccat tgtatttaaa aaaaaaaaaa aaaaaa 1536 18 400 PRT Glycine max 18Ala Arg Gly Lys Met Ser Val Ser His Phe Ala Val Leu Ser Ser Cys 1 5 1015 Cys Cys Pro Arg Leu Ala Pro Ser Leu Ser Arg Ala Ser Thr Leu Arg 20 2530 Ser Arg Ile Arg Cys Cys Thr Thr Leu Thr Ala Thr Ser Ser Glu Thr 35 4045 Pro Thr Pro Thr Phe Val Lys Lys Arg Val Val Ser Gly Val Gln Pro 50 5560 Thr Gly Ser Ile His Leu Gly Asn Tyr Phe Gly Ala Ile Lys Asn Trp 65 7075 80 Val Ala Leu Gln Asn Val Tyr Asp Thr Leu Phe Phe Ile Val Asp Leu 8590 95 His Ala Ile Thr Leu Pro Tyr Asp Thr Gln Gln Leu Ser Lys Ala Thr100 105 110 Arg Ser Thr Ala Ala Ile Tyr Leu Ala Cys Gly Val Asp Pro SerLys 115 120 125 Ala Ser Val Phe Val Gln Ser His Val Arg Ala His Val GluLeu Met 130 135 140 Trp Leu Leu Ser Ser Thr Thr Pro Ile Gly Trp Leu AsnLys Met Ile 145 150 155 160 Gln Phe Lys Glu Lys Ser Arg Lys Ala Gly AspGlu Glu Val Gly Val 165 170 175 Ala Leu Leu Thr Tyr Pro Val Leu Met AlaSer Asp Ile Leu Leu Tyr 180 185 190 Gln Ser Asp Phe Val Pro Val Gly GluAsp Gln Lys Gln His Leu Glu 195 200 205 Leu Thr Arg Asp Leu Ala Glu ArgVal Asn Asn Leu Tyr Gly Gly Arg 210 215 220 Lys Trp Lys Lys Leu Gly GlyTyr Asp Ser Arg Gly Gly Thr Ile Phe 225 230 235 240 Lys Val Pro Glu ProLeu Ile Pro Pro Ala Gly Ala Arg Ile Met Ser 245 250 255 Leu Thr Asp GlyLeu Ser Lys Met Ser Lys Ser Ala Pro Ser Asp Gln 260 265 270 Ser Arg IleAsn Ile Leu Asp Pro Lys Asp Leu Ile Ala Asn Lys Ile 275 280 285 Lys ArgCys Lys Thr Asp Ser Phe Pro Gly Leu Glu Phe Asp Asn Ser 290 295 300 GluArg Pro Glu Cys Asn Asn Leu Val Ser Ile Tyr Gln Leu Ile Ser 305 310 315320 Gly Lys Thr Lys Glu Glu Val Val Gln Glu Cys Gln Asn Met Asn Trp 325330 335 Gly Thr Phe Lys Pro Leu Leu Thr Asp Ala Leu Ile Asp His Leu His340 345 350 Pro Ile Gln Val Arg Tyr Glu Glu Ile Met Ser Asp Ser Gly TyrLeu 355 360 365 Asp Gly Val Leu Ala Gln Gly Ala Arg Asn Ala Ala Asp IleAla Asp 370 375 380 Ser Thr Leu Asn Asn Ile Tyr Gln Ala Met Gly Phe PheLys Arg Gln 385 390 395 400 19 725 DNA Triticum aestivum 19 ctcgtgccgaattcggcacg aggcggttca ttatttaagg ttcctgaagc ccttatccct 60 ccagcaggggcccgtgtgat gtccttaact gatggcctct ccaagatgtc gaagtctgct 120 ccttcagatttgtctcgcat taaccttctt gacccaaatg atgtgattgt gaacaaaatc 180 aaacgctgcaaaactgactc gctccctggc ttggaattcg acaacccaga gaggccggaa 240 tgcaaaaatcttctctcagt ctaccagatc atcactggaa aaacgaaaga ggaagttgtt 300 agtgaatgccaagatatgaa ctgggggacg ttcaaggtta cccttacgga tgccttaatt 360 gatcatctgcaacctattca ggttcgatac gaggagatca tgtctgatcc aggttatttg 420 gacaatgttctgctaaatgg ggcagggaaa gcttctgaga tagcagacgc caccctcaac 480 aacgtctaccaagccatggg tttcttgcgc agatagcata tgtagaacat tttttataac 540 tgcacaatgctagttttgca cttgttggcc tttctgctag tggtactgat aagcgttttg 600 tttgatatgcttggattagc cttttgttcc tggttattat ggacactgtt aataggtatt 660 aaaaggattatttactgaaa aaaaaaaaaa aaaaaaaaaa attaaaaggg ggcgcgcgta 720 ccata 725 20171 PRT Triticum aestivum 20 Leu Val Pro Asn Ser Ala Arg Gly Gly Ser LeuPhe Lys Val Pro Glu 1 5 10 15 Ala Leu Ile Pro Pro Ala Gly Ala Arg ValMet Ser Leu Thr Asp Gly 20 25 30 Leu Ser Lys Met Ser Lys Ser Ala Pro SerAsp Leu Ser Arg Ile Asn 35 40 45 Leu Leu Asp Pro Asn Asp Val Ile Val AsnLys Ile Lys Arg Cys Lys 50 55 60 Thr Asp Ser Leu Pro Gly Leu Glu Phe AspAsn Pro Glu Arg Pro Glu 65 70 75 80 Cys Lys Asn Leu Leu Ser Val Tyr GlnIle Ile Thr Gly Lys Thr Lys 85 90 95 Glu Glu Val Val Ser Glu Cys Gln AspMet Asn Trp Gly Thr Phe Lys 100 105 110 Val Thr Leu Thr Asp Ala Leu IleAsp His Leu Gln Pro Ile Gln Val 115 120 125 Arg Tyr Glu Glu Ile Met SerAsp Pro Gly Tyr Leu Asp Asn Val Leu 130 135 140 Leu Asn Gly Ala Gly LysAla Ser Glu Ile Ala Asp Ala Thr Leu Asn 145 150 155 160 Asn Val Tyr GlnAla Met Gly Phe Leu Arg Arg 165 170 21 1062 DNA Zea mays 21 gcacgagggacatcacgctg ctggatttcc tgagagaggt gggccgtttt gcacgcgtgg 60 gtacaatgatcgccaaggag agcgtcaaga agcgtcttgc gtcggaagac gggatgagct 120 acaccgagtttacctaccag ctgctgcagg gctacgactt cctttacatg ttcaagaata 180 tgggtgtcaatgtgcagatc gggggcagcg atcagtgggg gaacatcaca gcgggaactg 240 agttgatcagaaaaatcttg caggttgaag gggcgcatgg actcacattc ccacttctgc 300 tgaagagcgacggtaccaaa tttggaaaga cggaggatgg ggcaatctgg ctctcttcga 360 agatgctttctccttacaag ttctatcagt acttctttgc ggtgccagac atcgatgtca 420 tcaggtttatgaagatcctg acgttcctga gcttggatga gattctggag ctagaagact 480 cgatgaagaagcctggctat gtgccaaaca ctgttcagaa gaggcttgca gaagaggtga 540 cgcgatttgttcatggcgag gagggattgg aggaggcatt gaaggcaacc gaggccttga 600 gacctggtgctcagacacaa ttggatgcac aaacaattga ggggatagca gatgatgtgc 660 cttcatgctctttagcttat gatcaagtgt tcaagtctcc acttattgat ttggctgttt 720 ccacaggtttgctcactagt aagtcagcag ttaagcggct tattaagcaa ggtggtctgt 780 acttgaataacgtgaggatt gatagtgagg ataagctggt tgaggaaggt gatatagttg 840 atgggaaggtgctcttgttg tctgctggaa agaagaacaa gatggttgtg aggatatctt 900 gactactcttatttgttctt tataacttat tttagccatt gaggagaaaa gtaacggtgt 960 tgtgtcttcaaaactcaaat gagctgtcta tgagcataca gattgttata ttggagaggt 1020 tgaacacacctttttttttg ctctaaaaaa aaaaaaaaaa aa 1062 22 299 PRT Zea mays 22 Thr ArgAsp Ile Thr Leu Leu Asp Phe Leu Arg Glu Val Gly Arg Phe 1 5 10 15 AlaArg Val Gly Thr Met Ile Ala Lys Glu Ser Val Lys Lys Arg Leu 20 25 30 AlaSer Glu Asp Gly Met Ser Tyr Thr Glu Phe Thr Tyr Gln Leu Leu 35 40 45 GlnGly Tyr Asp Phe Leu Tyr Met Phe Lys Asn Met Gly Val Asn Val 50 55 60 GlnIle Gly Gly Ser Asp Gln Trp Gly Asn Ile Thr Ala Gly Thr Glu 65 70 75 80Leu Ile Arg Lys Ile Leu Gln Val Glu Gly Ala His Gly Leu Thr Phe 85 90 95Pro Leu Leu Leu Lys Ser Asp Gly Thr Lys Phe Gly Lys Thr Glu Asp 100 105110 Gly Ala Ile Trp Leu Ser Ser Lys Met Leu Ser Pro Tyr Lys Phe Tyr 115120 125 Gln Tyr Phe Phe Ala Val Pro Asp Ile Asp Val Ile Arg Phe Met Lys130 135 140 Ile Leu Thr Phe Leu Ser Leu Asp Glu Ile Leu Glu Leu Glu AspSer 145 150 155 160 Met Lys Lys Pro Gly Tyr Val Pro Asn Thr Val Gln LysArg Leu Ala 165 170 175 Glu Glu Val Thr Arg Phe Val His Gly Glu Glu GlyLeu Glu Glu Ala 180 185 190 Leu Lys Ala Thr Glu Ala Leu Arg Pro Gly AlaGln Thr Gln Leu Asp 195 200 205 Ala Gln Thr Ile Glu Gly Ile Ala Asp AspVal Pro Ser Cys Ser Leu 210 215 220 Ala Tyr Asp Gln Val Phe Lys Ser ProLeu Ile Asp Leu Ala Val Ser 225 230 235 240 Thr Gly Leu Leu Thr Ser LysSer Ala Val Lys Arg Leu Ile Lys Gln 245 250 255 Gly Gly Leu Tyr Leu AsnAsn Val Arg Ile Asp Ser Glu Asp Lys Leu 260 265 270 Val Glu Glu Gly AspIle Val Asp Gly Lys Val Leu Leu Leu Ser Ala 275 280 285 Gly Lys Lys AsnLys Met Val Val Arg Ile Ser 290 295 23 346 PRT Drosophila melanogaster23 Met Val Asp Lys Val Ala Asn Gly Val Ser Lys Lys Gly Ala Lys Lys 1 510 15 Ala Lys Ala Ala Lys Lys Ala Lys Ala Asn Ala Ser Thr Ala Ala Ala 2025 30 Asn Asn Ser Gly Gly Asp Ser Ala Asp His Ala Ala Gly Arg Tyr Gly 3540 45 Ser Met Ser Lys Asp Lys Arg Ser Arg Asn Val Val Ser Ser Gly Val 5055 60 Gly Lys Gly Val Trp Val Arg Gly Arg Val His Thr Ser Arg Ala Lys 6570 75 80 Gly Lys Cys Arg Ser Ser Thr Val Cys Ala Val Gly Asp Val Ser Lys85 90 95 Met Val Lys Ala Gly Asn Lys Ser Asp Ala Lys Val Ala Val Ser Ser100 105 110 Lys Ser Cys Thr Ser Ser Val Val Ser Ala Lys Ala Asp Ala SerArg 115 120 125 Asn Ala Asp Asp Ala Gly Asn Arg Val Asn Asp Thr Arg AspAsn Arg 130 135 140 Val Asp Arg Thr Ala Asn Ala Arg Ala Gly Val Cys ArgArg Asp Thr 145 150 155 160 Gly Thr His Thr Lys Ser Ala Ala Ser Gly GlyAla Asn Val Thr Val 165 170 175 Ser Tyr Lys Asp Ser Ala Tyr Ala Ser TyrLys Met Ala Ala Ala Asp 180 185 190 Asp Lys Val Tyr Thr Val Gly Ala ValArg Ala Asp Ser Asn Thr His 195 200 205 Arg His Thr Val Gly Asp Met AlaLys Tyr His Tyr His Val His Thr 210 215 220 Gly Asn Thr Thr Ser Lys GlyArg Asp Lys Tyr Ala Lys Ser Val Gly 225 230 235 240 Tyr Lys Val Asp AlaLys Ala Asp Gly Val Ala Met Arg Ala Gly Val 245 250 255 Thr Gly Asp AspSer Thr Asn Lys Gly Arg Val Lys Ala Lys Tyr Asp 260 265 270 Thr Asp TyrAsp Lys Ala Arg Tyr Thr Met Asp Asn Asn Val Tyr Ser 275 280 285 Asn SerTyr Asp Met Met Arg Gly Ser Gly Ala Arg His Asp Tyr Arg 290 295 300 AlaLys His His Gly Asp Thr Ser Lys Ala Ala Tyr Ser Arg Tyr Gly 305 310 315320 Cys His Ala Gly Gly Gly Gly Met Arg Val Val Met Tyr Gly Asp Asn 325330 335 Arg Lys Thr Ser Met Arg Asp Lys Arg Thr 340 345 24 501 PRTRattus norvegicus 24 Met Pro Ser Ala Asn Ala Ser Arg Lys Gly Gln Glu LysPro Arg Glu 1 5 10 15 Ile Val Asp Ala Ala Glu Asp Tyr Ala Lys Glu ArgTyr Gly Val Ser 20 25 30 Ser Met Ile Gln Ser Gln Glu Lys Pro Asp Arg ValLeu Val Arg Val 35 40 45 Lys Asp Leu Thr Val Gln Lys Ala Asp Glu Val ValTrp Val Arg Ala 50 55 60 Arg Val His Thr Ser Arg Ala Lys Gly Lys Gln CysPhe Leu Val Leu 65 70 75 80 Arg Gln Gln Gln Phe Asn Val Gln Ala Leu ValAla Val Gly Asp His 85 90 95 Ala Ser Lys Gln Met Val Lys Phe Ala Ala AsnIle Asn Lys Glu Ser 100 105 110 Ile Ile Asp Val Glu Gly Ile Val Arg LysVal Asn Gln Lys Ile Gly 115 120 125 Ser Cys Thr Gln Gln Asp Val Glu LeuHis Val Gln Lys Ile Tyr Val 130 135 140 Ile Ser Leu Ala Glu Pro Arg LeuPro Leu Gln Leu Asp Asp Ala Ile 145 150 155 160 Arg Pro Glu Val Glu GlyGlu Glu Asp Gly Arg Ala Thr Val Asn Gln 165 170 175 Asp Thr Arg Leu AspAsn Arg Ile Ile Asp Leu Arg Thr Ser Thr Ser 180 185 190 Gln Ala Ile PheHis Leu Gln Ser Gly Ile Cys His Leu Phe Arg Glu 195 200 205 Thr Leu IleAsn Lys Gly Phe Val Glu Ile Gln Thr Pro Lys Ile Ile 210 215 220 Ser AlaAla Ser Glu Gly Gly Ala Asn Val Phe Thr Val Ser Tyr Phe 225 230 235 240Lys Ser Asn Ala Tyr Leu Ala Gln Ser Pro Gln Leu Tyr Lys Gln Met 245 250255 Cys Ile Cys Ala Asp Phe Glu Lys Val Phe Cys Ile Gly Pro Val Phe 260265 270 Arg Ala Glu Asp Ser Asn Thr His Arg His Leu Thr Glu Phe Val Gly275 280 285 Leu Asp Ile Glu Met Ala Phe Asn Tyr His Tyr His Glu Val ValGlu 290 295 300 Glu Ile Ala Asp Thr Leu Val Gln Ile Phe Lys Gly Leu GlnGlu Arg 305 310 315 320 Phe Gln Thr Glu Ile Gln Thr Val Asn Lys Gln PhePro Cys Glu Pro 325 330 335 Phe Lys Phe Leu Glu Pro Thr Leu Arg Leu GluTyr Cys Glu Ala Leu 340 345 350 Ala Met Leu Arg Glu Ala Gly Val Glu MetAsp Asp Glu Glu Asp Leu 355 360 365 Ser Thr Pro Asn Glu Lys Leu Leu GlyArg Leu Val Lys Glu Lys Tyr 370 375 380 Asp Thr Asp Phe Tyr Val Leu AspLys Tyr Pro Leu Ala Val Arg Pro 385 390 395 400 Phe Tyr Thr Met Pro AspPro Arg Asn Pro Lys Gln Ser Asn Ser Tyr 405 410 415 Asp Met Phe Met ArgGly Glu Glu Ile Leu Ser Gly Ala Gln Arg Ile 420 425 430 His Asp Pro GlnLeu Leu Thr Glu Arg Ala Leu His His Gly Ile Asp 435 440 445 Leu Glu LysIle Lys Ala Tyr Ile Asp Ser Phe Arg Phe Gly Ala Pro 450 455 460 Pro HisAla Gly Gly Gly Ile Gly Leu Glu Arg Val Thr Met Leu Phe 465 470 475 480Leu Gly Leu His Asn Val Arg Gln Thr Ser Met Phe Pro Arg Asp Pro 485 490495 Lys Arg Leu Thr Pro 500 25 500 PRT Homo sapiens 25 Met Pro Ser AlaThr Gln Arg Lys Ser Gln Glu Lys Pro Arg Glu Ile 1 5 10 15 Met Asp AlaAla Glu Asp Tyr Ala Lys Glu Arg Tyr Gly Ile Ser Ser 20 25 30 Met Ile GlnSer Gln Glu Lys Pro Asp Arg Val Leu Val Arg Val Arg 35 40 45 Asp Leu ThrIle Gln Lys Ala Asp Glu Val Val Trp Val Arg Ala Arg 50 55 60 Val His ThrSer Arg Ala Lys Gly Lys Gln Cys Phe Leu Val Leu Arg 65 70 75 80 Gln GlnGln Phe Asn Val Gln Ala Leu Val Ala Val Gly Asp His Ala 85 90 95 Ser LysGln Met Val Lys Phe Ala Ala Asn Ile Asn Lys Glu Ser Ile 100 105 110 ValAsp Val Glu Gly Val Val Arg Lys Val Asn Gln Lys Ile Gly Ser 115 120 125Cys Thr Gln Gln Asp Val Glu Leu His Val Gln Lys Ile Tyr Val Ile 130 135140 Ser Leu Ala Glu Pro Arg Leu Pro Leu Gln Leu Asp Asp Ala Val Arg 145150 155 160 Pro Glu Gln Glu Gly Glu Glu Glu Gly Arg Ala Thr Val Asn GlnAsp 165 170 175 Thr Arg Leu Asp Asn Arg Val Ile Asp Leu Arg Thr Ser ThrSer Gln 180 185 190 Ala Val Phe Arg Leu Gln Ser Gly Ile Cys His Leu PheArg Glu Thr 195 200 205 Leu Ile Asn Lys Gly Phe Val Glu Ile Gln Thr ProLys Ile Ile Ser 210 215 220 Ala Ala Ser Glu Gly Gly Ala Asn Val Phe ThrVal Ser Tyr Phe Lys 225 230 235 240 Asn Asn Ala Tyr Leu Ala Gln Ser ProGln Leu Tyr Lys Gln Met Cys 245 250 255 Ile Cys Ala Asp Phe Glu Lys ValPhe Ser Ile Gly Pro Val Phe Arg 260 265 270 Ala Glu Asp Ser Asn Thr HisArg His Leu Thr Glu Phe Val Gly Leu 275 280 285 Asp Ile Glu Met Ala PheAsn Tyr His Tyr His Glu Val Met Glu Glu 290 295 300 Ile Ala Asp Thr MetVal Gln Ile Phe Lys Gly Leu Gln Glu Arg Phe 305 310 315 320 Gln Thr GluIle Gln Thr Val Asn Lys Gln Phe Pro Cys Glu Pro Phe 325 330 335 Lys PheLeu Glu Pro Thr Leu Arg Leu Glu Tyr Cys Glu Ala Leu Ala 340 345 350 MetLeu Arg Glu Ala Gly Val Glu Met Gly Asp Glu Asp Asp Leu Ser 355 360 365Thr Pro Asn Glu Lys Leu Leu Gly His Leu Val Lys Glu Lys Tyr Asp 370 375380 Thr Asp Phe Tyr Ile Leu Asp Lys Tyr Pro Leu Ala Val Arg Pro Phe 385390 395 400 Tyr Thr Met Pro Asp Pro Arg Asn Pro Lys Gln Ser Lys Ser TyrAsp 405 410 415 Met Phe Met Arg Gly Glu Glu Ile Leu Ser Gly Ala Gln ArgIle His 420 425 430 Asp Pro Gln Leu Leu Thr Glu Arg Ala Leu His His GlyAsn Asp Leu 435 440 445 Glu Lys Ile Lys Ala Tyr Ile Asp Ser Phe Arg PheGly Ala Pro Pro 450 455 460 His Ala Gly Gly Gly Ile Gly Leu Glu Arg ValThr Met Leu Phe Leu 465 470 475 480 Gly Leu His Asn Val Arg Gln Thr SerMet Phe Pro Arg Asp Pro Lys 485 490 495 Arg Leu Thr Pro 500 26 459 PRTHaemophilus influenzae Rd 26 Met Leu Lys Ile Phe Asn Thr Leu Thr Arg GluLys Glu Ile Phe Lys 1 5 10 15 Pro Ile His Glu Asn Lys Val Gly Met TyrVal Cys Gly Val Thr Val 20 25 30 Tyr Asp Leu Cys His Ile Gly His Gly ArgThr Phe Val Cys Phe Asp 35 40 45 Val Ile Ala Arg Tyr Leu Arg Ser Leu GlyTyr Asp Leu Thr Tyr Val 50 55 60 Arg Asn Ile Thr Asp Val Asp Asp Lys IleIle Lys Arg Ala Leu Glu 65 70 75 80 Asn Lys Glu Thr Cys Asp Gln Leu ValAsp Arg Met Val Gln Glu Met 85 90 95 Tyr Lys Asp Phe Asp Ala Leu Asn ValLeu Arg Pro Asp Phe Glu Pro 100 105 110 Arg Ala Thr His His Ile Pro GluIle Ile Glu Ile Val Glu Lys Leu 115 120 125 Ile Lys Arg Gly His Ala TyrVal Ala Asp Asn Gly Asp Val Met Phe 130 135 140 Asp Val Glu Ser Phe LysGlu Tyr Gly Lys Leu Ser Arg Gln Asp Leu 145 150 155 160 Glu Gln Leu GlnAla Gly Ala Arg Ile Glu Ile Asn Glu Ile Lys Lys 165 170 175 Asn Pro MetAsp Phe Val Leu Trp Lys Met Ser Lys Glu Asn Glu Pro 180 185 190 Ser TrpAla Ser Pro Trp Gly Ala Gly Arg Pro Gly Trp His Ile Glu 195 200 205 CysSer Ala Met Asn Cys Lys Gln Leu Gly Glu Tyr Phe Asp Ile His 210 215 220Gly Gly Gly Ser Asp Leu Met Phe Pro His His Glu Asn Glu Ile Ala 225 230235 240 Gln Ser Cys Cys Ala His Gly Gly Gln Tyr Val Asn Tyr Trp Ile His245 250 255 Ser Gly Met Ile Met Val Asp Lys Glu Lys Met Ser Lys Ser LeuGly 260 265 270 Asn Phe Phe Thr Ile Arg Asp Val Leu Asn His Tyr Asn AlaGlu Ala 275 280 285 Val Arg Tyr Phe Leu Leu Thr Ala His Tyr Arg Ser GlnLeu Asn Tyr 290 295 300 Ser Glu Glu Asn Leu Asn Leu Ala Gln Gly Ala LeuGlu Arg Leu Tyr 305 310 315 320 Thr Ala Leu Arg Gly Thr Asp Gln Ser AlaVal Ala Phe Gly Gly Glu 325 330 335 Asn Phe Val Ala Thr Phe Arg Glu AlaMet Asp Asp Asp Phe Asn Thr 340 345 350 Pro Asn Ala Leu Ser Val Leu PheGlu Met Ala Arg Glu Ile Asn Lys 355 360 365 Leu Lys Thr Glu Asp Val GluLys Ala Asn Gly Leu Ala Ala Arg Leu 370 375 380 Arg Glu Leu Gly Ala IleLeu Gly Leu Leu Gln Gln Glu Pro Glu Lys 385 390 395 400 Phe Leu Gln AlaGly Ser Asn Asp Asp Glu Val Ala Lys Ile Glu Ala 405 410 415 Leu Ile LysGln Arg Asn Glu Ala Arg Thr Ala Lys Asp Trp Ser Ala 420 425 430 Ala AspSer Ala Arg Asn Glu Leu Thr Ala Met Gly Ile Val Leu Glu 435 440 445 AspGly Pro Asn Gly Thr Thr Trp Arg Lys Gln 450 455 27 461 PRT Escherichiacoli 27 Met Leu Lys Ile Phe Asn Thr Leu Thr Arg Gln Lys Glu Glu Phe Lys1 5 10 15 Pro Ile His Ala Gly Glu Val Gly Met Tyr Val Cys Gly Ile ThrVal 20 25 30 Tyr Asp Leu Cys His Ile Gly His Gly Arg Thr Phe Val Ala PheAsp 35 40 45 Val Val Ala Arg Tyr Leu Arg Phe Leu Gly Tyr Lys Leu Lys TyrVal 50 55 60 Arg Asn Ile Thr Asp Ile Asp Asp Lys Ile Ile Lys Arg Ala AsnGlu 65 70 75 80 Asn Gly Glu Ser Phe Val Ala Met Val Asp Arg Met Ile AlaGlu Met 85 90 95 His Lys Asp Phe Asp Ala Leu Asn Ile Leu Arg Pro Asp MetGlu Pro 100 105 110 Arg Ala Thr His His Ile Ala Glu Ile Ile Glu Leu ThrGlu Gln Leu 115 120 125 Ile Ala Lys Gly His Ala Tyr Val Ala Asp Asn GlyAsp Val Met Phe 130 135 140 Asp Val Pro Thr Asp Pro Thr Tyr Gly Val LeuSer Arg Gln Asp Leu 145 150 155 160 Asp Gln Leu Gln Ala Gly Ala Arg ValAsp Val Val Asp Asp Lys Arg 165 170 175 Asn Pro Met Asp Phe Val Leu TrpLys Met Ser Lys Glu Gly Glu Pro 180 185 190 Ser Trp Pro Ser Pro Trp GlyAla Gly Arg Pro Gly Trp His Ile Glu 195 200 205 Cys Ser Ala Met Asn CysLys Gln Leu Gly Asn His Phe Asp Ile His 210 215 220 Gly Gly Gly Ser AspLeu Met Phe Pro His His Glu Asn Glu Ile Ala 225 230 235 240 Gln Ser ThrCys Ala His Asp Gly Gln Tyr Val Asn Tyr Trp Met His 245 250 255 Ser GlyMet Val Met Val Asp Arg Glu Lys Met Ser Lys Ser Leu Gly 260 265 270 AsnPhe Phe Thr Val Arg Asp Val Leu Lys Tyr Tyr Asp Ala Glu Thr 275 280 285Val Arg Tyr Phe Leu Met Ser Gly His Tyr Arg Ser Gln Leu Asn Tyr 290 295300 Ser Glu Glu Asn Leu Lys Gln Ala Arg Ala Ala Val Glu Arg Leu Tyr 305310 315 320 Thr Ala Leu Arg Gly Thr Asp Lys Thr Val Ala Pro Ala Gly GlyGlu 325 330 335 Ala Phe Glu Ala Arg Phe Ile Glu Ala Met Asp Asp Asp PheAsn Thr 340 345 350 Pro Glu Ala Tyr Ser Val Leu Phe Asp Met Ala Arg GluVal Asn Arg 355 360 365 Leu Lys Ala Glu Asp Met Ala Ala Ala Asn Ala MetAla Ser His Leu 370 375 380 Arg Lys Leu Ser Ala Val Leu Gly Leu Leu GluGln Glu Pro Glu Ala 385 390 395 400 Phe Leu Gln Ser Gly Ala Gln Ala AspAsp Ser Glu Val Ala Glu Ile 405 410 415 Glu Ala Leu Ile Gln Gln Arg LeuAsp Ala Arg Lys Ala Lys Asp Trp 420 425 430 Ala Ala Ala Asp Ala Ala ArgAsp Arg Leu Asn Glu Met Gly Ile Val 435 440 445 Leu Glu Asp Gly Pro GlnGly Thr Thr Trp Arg Arg Lys 450 455 460 28 377 PRT Synechocystis sp. 28Met Lys Asn Cys Glu Asn Asp His Arg Phe Thr Thr Val Ser Ser Gly 1 5 1015 Lys Ala Trp Gly Gln Leu His Arg Phe Pro Ser Leu Ile Lys Phe Asn 20 2530 Phe Ala His Arg Ser Thr Thr Ala Met Asp Lys Pro Arg Ile Leu Ser 35 4045 Gly Val Gln Pro Thr Gly Asn Leu His Leu Gly Asn Tyr Leu Gly Ala 50 5560 Ile Arg Ser Trp Val Glu Gln Gln Gln His Tyr Asp Asn Phe Phe Cys 65 7075 80 Val Val Asp Leu His Ala Ile Thr Val Pro His Asn Pro Gln Thr Leu 8590 95 Ala Gln Asp Thr Leu Thr Ile Ala Ala Leu Tyr Leu Ala Cys Gly Ile100 105 110 Asp Leu Gln Tyr Ser Thr Ile Phe Val Gln Ser His Val Ala AlaHis 115 120 125 Ser Glu Leu Ala Trp Leu Leu Asn Cys Val Thr Pro Leu AsnTrp Leu 130 135 140 Glu Arg Met Ile Gln Phe Lys Glu Lys Ala Val Lys GlnGly Glu Asn 145 150 155 160 Val Ser Val Gly Leu Leu Asp Tyr Pro Val LeuMet Ala Ala Asp Ile 165 170 175 Leu Leu Tyr Asp Ala Asp Lys Val Pro ValGly Glu Asp Gln Lys Gln 180 185 190 His Leu Glu Leu Thr Arg Asp Ile ValIle Arg Ile Asn Asp Lys Phe 195 200 205 Gly Arg Glu Asp Ala Pro Val LeuLys Leu Pro Glu Pro Leu Ile Arg 210 215 220 Lys Glu Gly Ala Arg Val MetSer Leu Ala Asp Gly Thr Lys Lys Met 225 230 235 240 Ser Lys Ser Asp GluSer Glu Leu Ser Arg Ile Asn Leu Leu Asp Pro 245 250 255 Pro Glu Met IleLys Lys Lys Val Lys Lys Cys Lys Thr Asp Pro Gln 260 265 270 Arg Gly LeuTrp Phe Asp Asp Pro Glu Arg Pro Glu Cys His Asn Leu 275 280 285 Leu ThrLeu Tyr Thr Leu Leu Ser Asn Gln Thr Lys Glu Ala Val Ala 290 295 300 GlnGlu Cys Ala Glu Met Gly Trp Gly Gln Phe Lys Pro Leu Leu Thr 305 310 315320 Glu Thr Ala Ile Ala Ala Leu Glu Pro Ile Gln Ala Lys Tyr Ala Glu 325330 335 Ile Leu Ala Asp Arg Gly Glu Leu Asp Arg Ile Ile Gln Ala Gly Asn340 345 350 Ala Lys Ala Ser Gln Thr Ala Gln Gln Thr Leu Ala Arg Val ArgAsp 355 360 365 Ala Leu Gly Phe Leu Ala Pro Pro Tyr 370 375 29 419 PRTBacillus caldotenax 29 Met Asp Leu Leu Ala Glu Leu Gln Trp Arg Gly LeuVal Asn Gln Thr 1 5 10 15 Thr Asp Glu Asp Gly Leu Arg Lys Leu Leu AsnGlu Glu Arg Val Thr 20 25 30 Leu Tyr Cys Gly Phe Asp Pro Thr Ala Asp SerLeu His Ile Gly Asn 35 40 45 Leu Ala Ala Ile Leu Thr Leu Arg Arg Phe GlnGln Ala Gly His Arg 50 55 60 Pro Ile Ala Leu Val Gly Gly Ala Thr Gly LeuIle Gly Asp Pro Ser 65 70 75 80 Gly Lys Lys Ser Glu Arg Thr Leu Asn AlaLys Glu Thr Val Glu Ala 85 90 95 Trp Ser Ala Arg Ile Lys Glu Gln Leu GlyArg Phe Leu Asp Phe Glu 100 105 110 Ala Asp Gly Asn Pro Ala Lys Ile LysAsn Asn Tyr Asp Trp Ile Gly 115 120 125 Pro Leu Asp Val Ile Thr Phe LeuArg Asp Val Gly Lys His Phe Ser 130 135 140 Val Asn Tyr Met Met Ala LysGlu Ser Val Gln Ser Arg Ile Glu Thr 145 150 155 160 Gly Ile Ser Phe ThrGlu Phe Ser Tyr Met Met Leu Gln Ala Tyr Asp 165 170 175 Phe Leu Arg LeuTyr Glu Thr Glu Gly Cys Arg Leu Gln Ile Gly Gly 180 185 190 Ser Asp GlnTrp Gly Asn Ile Thr Ala Gly Leu Glu Leu Ile Arg Lys 195 200 205 Thr LysGly Glu Ala Arg Ala Phe Gly Leu Thr Ile Pro Leu Val Thr 210 215 220 LysAla Asp Gly Thr Lys Phe Gly Lys Thr Glu Ser Gly Thr Ile Trp 225 230 235240 Leu Asp Lys Glu Lys Thr Ser Pro Tyr Glu Phe Tyr Gln Phe Trp Ile 245250 255 Asn Thr Asp Asp Arg Asp Val Ile Arg Tyr Leu Lys Tyr Phe Thr Phe260 265 270 Leu Ser Lys Glu Glu Ile Glu Ala Leu Glu Gln Glu Leu Arg GluAla 275 280 285 Pro Glu Lys Arg Ala Ala Gln Lys Ala Leu Ala Glu Glu ValThr Lys 290 295 300 Leu Val His Gly Glu Glu Ala Leu Arg Gln Ala Ile ArgIle Ser Glu 305 310 315 320 Ala Leu Phe Ser Gly Asp Ile Ala Asn Leu ThrAla Ala Glu Ile Glu 325 330 335 Gln Gly Phe Lys Asp Val Pro Ser Phe ValHis Glu Gly Gly Asp Val 340 345 350 Pro Leu Val Glu Leu Leu Val Ser AlaGly Ile Ser Pro Ser Lys Arg 355 360 365 Gln Ala Arg Glu Asp Ile Gln AsnGly Ala Ile Tyr Val Asn Gly Glu 370 375 380 Arg Leu Gln Asp Val Gly AlaIle Leu Thr Ala Glu His Arg Leu Glu 385 390 395 400 Gly Arg Phe Thr ValIle Arg Arg Gly Lys Lys Lys Tyr Tyr Leu Ile 405 410 415 Arg Tyr Ala

What is claimed is:
 1. An isolated nucleic acid fragment encoding anaspartyl-tRNA synthetase comprising a member selected from the groupconsisting of: (a) an isolated nucleic acid fragment encoding an aminoacid sequence that is at least 80% identical to the amino acid sequenceset forth in a member selected from the group consisting of SEQ ID NO:2, 4, 6 and 8; (b) an isolated nucleic acid fragment that iscomplementary to (a).
 2. The isolated nucleic acid fragment of claim 1wherein nucleic acid fragment is a functional RNA.
 3. The isolatednucleic acid fragment of claim 1 wherein the nucleotide sequence of thefragment comprises the sequence set forth in a member selected from thegroup consisting of SEQ ID NO: 1, 3, 5 and
 7. 4. A chimeric genecomprising the nucleic acid fragment of claim 1 operably linked tosuitable regulatory sequences.
 5. A transformed host cell comprising thechimeric gene of claim
 4. 6. An aspartyl-tRNA synthetase polypeptidecomprising all or a substantial portion of the amino acid sequence setforth in a member selected from the group consisting of SEQ ID NO: 2.,4, 6 and 8
 7. An isolated nucleic acid fragment encoding acysteinyl-tRNA synthetase comprising a member selected from the groupconsisting of: (a) an isolated nucleic acid fragment encoding an aminoacid sequence that is at least 80% identical to the amino acid sequenceset forth in a member selected from the group consisting of SEQ ID NO:10, 12 and 14; (b) an isolated nucleic acid fragment that iscomplementary to (a).
 8. The isolated nucleic acid fragment of claim 7wherein nucleic acid fragment is a functional RNA.
 9. The isolatednucleic acid fragment of claim 7 wherein the nucleotide sequence of thefragment comprises the sequence set forth in a member selected from thegroup consisting of SEQ ID NO: 9, 11 and
 13. 10. A chimeric genecomprising the nucleic acid fragment of claim 7 operably linked tosuitable regulatory sequences.
 11. A transformed host cell comprisingthe chimeric gene of claim
 10. 12. A cysteinyl-tRNA synthetasepolypeptide comprising all or a substantial portion of the amino acidsequence set forth in a member selected from the group consisting of SEQID NO: 10, 12 and
 14. 13. An isolated nucleic acid fragment encoding atryptophanyl-tRNA synthetase comprising a member selected from the groupconsisting of: (a) an isolated nucleic acid fragment encoding an aminoacid sequence that is at least 80% identical to the amino acid sequenceset forth in a member selected from the group consisting of SEQ ID NO:16, 18 and 20; (b) an isolated nucleic acid fragment that iscomplementary to (a).
 14. The isolated nucleic acid fragment of claim 13wherein nucleic acid fragment is a functional RNA.
 15. The isolatednucleic acid fragment of claim 13 wherein the nucleotide sequence of thefragment comprises the sequence set forth in a member selected from thegroup consisting of SEQ ID NO: 15, 17 and
 19. 16. A chimeric genecomprising the nucleic acid fragment of claim 13 operably linked tosuitable regulatory sequences.
 17. A transformed host cell comprisingthe chimeric gene of claim
 16. 18. A tryptophanyl-tRNA synthetasepolypeptide comprising all or a substantial portion of the amino acidsequence set forth in a member selected from the group consisting of SEQID NO: 16, 18 and
 20. 19. An isolated nucleic acid fragment encoding atyrosyl-tRNA synthetase comprising a member selected from the groupconsisting of: (a) an isolated nucleic acid fragment encoding an aminoacid sequence that is at least 80% identical to the amino acid sequenceset forth in SEQ ID NO: 22; (b) an isolated nucleic acid fragment thatis complementary to (a).
 20. The isolated nucleic acid fragment of claim19 wherein nucleic acid fragment is a functional RNA.
 21. The isolatednucleic acid fragment of claim 19 wherein the nucleotide sequence of thefragment comprises the sequence set forth in SEQ ID NO: 2
 1. 22. Achimeric gene comprising the nucleic acid fragment of claim 19 operablylinked to suitable regulatory sequences.
 23. A transformed host cellcomprising the chimeric gene of claim
 22. 24. A tyrosyl-tRNA synthetasepolypeptide comprising all or a substantial portion of the amino acidsequence set forth in SEQ ID NO:
 22. 25. A method of altering the levelof expression of an aminoacyl-tRNA synthetase in a host cell comprising:(a) transforming a host cell with the chimeric gene of any of claims 4,10, 16 and 22; and (b) growing the transformed host cell produced instep (a) under conditions that are suitable for expression of thechimeric gene wherein expression of the chimeric gene results inproduction of altered levels of an aminoacyl-tRNA synthetase in thetransformed host cell.
 26. A method of obtaining a nucleic acid fragmentencoding all or a substantial portion of the amino acid sequenceencoding an aminoacyl-tRNA synthetase comprising: (a) probing a cDNA orgenomic library with the nucleic acid fragment of any of claims 1, 7,13and 19; (b) identifying a DNA clone that hybridizes with the nucleicacid fragment of any of claims 1, 7, 13 and 19; (c) isolating the DNAclone identified in step (b); and (d) sequencing the cDNA or genomicfragment that comprises the clone isolated in step (c) wherein thesequenced nucleic acid fragment encodes all or a substantial portion ofthe amino acid sequence encoding an aminoacyl-tRNA synthetase.
 27. Amethod of obtaining a nucleic acid fragment encoding a substantialportion of an amino acid sequence encoding an aminoacyl-tRNA synthetasecomprising: (a) synthesizing an oligonucleotide primer corresponding toa portion of the sequence set forth in any of SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 17, 19 and 21; and (b) amplifying a cDNA insert present in acloning vector using the oligonucleotide primer of step (a) and a primerrepresenting sequences of the cloning vector wherein the amplifiednucleic acid fragment encodes a substantial portion of an amino acidsequence encoding an aminoacyl-tRNA synthetase.
 28. The product of themethod of claim
 26. 29. The product of the method of claim
 27. 30. Amethod for evaluating at least one compound for its ability to inhibitthe activity of an aminoacyl-tRNA synthetase, the method comprising thesteps of: (a) transforming a host cell with a chimeric gene comprising anucleic acid fragment encoding an aminoacyl-tRNA synthetase, operablylinked to suitable regulatory sequences; (b) growing the transformedhost cell under conditions that are suitable for expression of thechimeric gene wherein expression of the chimeric gene results inproduction of the aminoacyl-tRNA synthetase encoded by the operablylinked nucleic acid fragment in the transformed host cell; (c)optionally purifying the aminoacyl-tRNA synthetase expressed by thetransformed host cell; (d) treating the aminoacyl-tRNA synthetase with acompound to be tested; and (e) comparing the activity of theaminoacyl-tRNA synthetase that has been treated with a test compound tothe activity of an untreated aminoacyl-tRNA synthetase, therebyselecting compounds with potential for inhibitory activity.